Microplastic Identification in Domestic Wastewater-Treating Constructed Wetlands and Its Potential Usage in a Circular Economy

Abstract

Mentions of microplastics (MPs) are increasingly frequent, for they are present in all environments, including wastewater. Knowing their possible harmful effects on the food chain, the fact that they appear in crops is concerning. The ways by which they are transported and stored, as well as their final destination, are still unclear. The issue of MPs in wastewater and how they are carried into agricultural crops are little-known facts. This study aims to evaluate whether horizontal subsurface flow wetlands with ornamental plants (Hippeastrum hybridum hort and Heliconia bihai marginata) can retain microplastics present in domestic wastewater while at the same time recirculating water for irrigation of the Phaseolus vulgaris crop. On average, the ornamental plants Hippeastrum hybridum hort and Heliconia bihai marginata removed contaminants such as COD, NH₄⁺, TN, NO₂⁻, TP, PO₄³⁻, and TSS, with an efficiency of 84% and 98%, respectively. The presence of MPs was identified via FTIR analysis and visual characterization in domestic wastewater, treated wastewater, and well water; the quality of the fruit for human consumption was determined using safety tests for Escherichia coli and Salmonella.

1. Introduction

The need for plastic products in our everyday activities has triggered an uncontrolled increase in their production and, as a consequence, also significant contamination of all ecosystems of the planet, both aquatic and terrestrial [1]. Combined environmental factors cause plastics to fragment and disintegrate, thus creating microplastics (MPs), classified as an emerging pollutant [2]. Microplastics are biomagnifying and bio-accumulative [3], being harmful to human health and causing carcinogenic diseases, neurotoxicity, and also affecting reproductive health [4]. They can cause DNA damage, cell damage, and inflammation as well [5]. Their presence in the environment is due to the difficulty of their disposal via physical, chemical, and wastewater treatment plant (WWTP) methods [6,7,8]. In recent years, there has been a growing interest in the identification of MPs, and they have been studied in aquatic fauna [9], air, soils [10], and in the atmosphere. Also, there is also acute interest in the mobility of these materials through rain [11], in groundwater, in wastewater [12] and in surface waters (rivers) [13], but despite this, the carrying, migration, and fate of microplastics are still unknown facts [14]. The presence of MPs in water is a current issue requiring prompt solutions considering that these particles have been found throughout the aquatic ecosystem, including wastewater [15]. The presence of MPs in water is of concern because they have the potential for absorbing or releasing contaminants from wastewater and introducing them into the food chain [16]. This problem is exacerbated by the practice of irrigating crops with untreated wastewater, a situation prevalent in Mexico, where 85% of crops receive this type of irrigation [17].

This has led some researchers to try to remove MPs from wastewater using traditional treatment plants (WWTPs), but the MPs remain undamaged in the water after treatment [18,19,20]. Other research work has focused on nature-based solutions, such as constructed wetlands (CWs), which have proven to be useful for cleaning industrial and domestic wastewater, even wastewater containing compounds such as ibuprofen and carbamazepine [21]. Given their removal features (physical, chemical, and biological), CWs were considered a possible alternative for successfully eliminating MPs, and according to some studies, CWs manage to remove MPs in domestic wastewater with an efficiency of up to 98.13% [22]. For waters in which concentrations of Polypropylene (PP) at 54.6%, polystyrene (PS) at 29.7%, Polyethylene Terephthalate (PET), and Vinyl Polychloride (PVC)—the most common polymers in domestic wastewater effluents—were found, the removal of MPs in CWs was higher than 73% in wetlands with anaerobic treatment [22], in which vegetation plays an important role [23,24,25]. While the concept of a circular economy has represented a valuable change with a focus on sustainability, the recirculation of water treated by CWs has been poorly documented [26]. Only the possibility of mitigating net water consumption by reusing it for agricultural irrigation, parks, gardens, and cleaning has been mentioned [27]. Subsurface flow CWs (SSCWs) were recently reported to retain MPs [28,29] with an efficiency of up to 98.13%, however, being placed as secondary or tertiary treatment [30,31]. The most commonly used flow type is horizontal subsurface [32], and flowering ornamental plants are also reported as key elements in the removal efficiency of common pollutants in wastewater [33].

In this context, the present study aims to address two objectives:

• To identify the presence of microplastics in domestic wastewater treated by horizontal subsurface flow constructed wetlands with ornamental plants using FTIR spectroscopy and visual characterization;
• To assess constructed wetland-treated wastewater reuse for Phaseolus vulgaris irrigation as a circular economy strategy.

2. Materials and Methods

The experiment was designed with a two-fold goal in mind, and the following stages, as described below, were implemented for achieving it:

• Stage 1. Selection of crop and study area.

Phaseolus vulgaris is a legume that is part of the three food groups of the Healthy Eating Plate (NOM-043-SSA2-2005) [34]. It is the third most important legume in terms of planted area in Mexico [30,35] and has a short vegetative cycle (three to four months from sowing to harvest). In addition, the climate, soil properties of the experimental area, overall coefficient (kg), and type of crop exposure requirement, as well as the distance between vines for planting seeds and the type of irrigation system, are conditions that must be considered for proper crop development.

Moreover, the selection of the crop allowed us to analyze the production of Phaseolus vulgaris in the Misantla area. Figure 1 shows the sowing capacity for this legume where it is grown; it grows well in the altitude and rainfall conditions of the area (based on an analysis from a high resolution, 5 m; the optimal slopes for this crop are between 200 and 1000 m above sea level, and the best rainfall amount is between 300 and 400 mm. Other general aspects of the area are also relevant and deserve to be described.

• Study area.

Misantla is one of the 212 municipalities comprising Veracruz State, Mexico, located in the mountainous region of the central part of the state, with an altitude of 380 m above sea level. Its climate is defined as warm–humid (tropical) with an average temperature of 27 °C. An area of 176.6 km² is dedicated to agriculture.

The study area has a mean annual precipitation of 2200 to 2280 mm. Analyses conducted with the DAVIS 6152 Vantage Pro2 weather station indicate that Misantla experiences temperature fluctuations ranging from 16 to 25–29 °C. The experimental area has temperatures between 21 and 22 °C. This temperature range is ideal for the cultivation of the Phaseolus vulgaris. The Agrifood and Fisheries Information Service (Servicio de Información Agroalimentaria y Pesquera, SIAP) indicates that this crop grows optimally between 10 and 27 °C. Peak biometric development and harvest yields are expected, considering that the experiment is carried out in the open and not under controlled conditions.

However, farmers in the area have resorted to unreliable alternatives to meet crop water requirements. Natural alternatives have been proposed for addressing this problem [36], such as constructed wetland (CW) systems, and a treated water recirculation system was adapted for watering the crop as part of this experiment.

• Stage 2. Setting up of the horizontal subsurface flow constructed wetland system (HSSF-CW).

Studies of constructed wetland systems with plants mention that Heliconia, Heliconia latispatha, Heliconia bihai marginata, Canna, Iris, and Alpinia purpurata have thicker root densities, with roots that are deep and distributed throughout the wetland system [37]. Horizontal flow and cell depth play an important role in the removal of common pollutants in domestic wastewater, hence the importance of properly controlling them; there are still no reports that indicate whether flow, roots, and/or climatic conditions can favor the removal of MPs [32,38,39,40,41,42]. In this regard, this work makes use of horizontal subsurface flow CWs conditioned with two types of ornamental plants, Hippeastrum hybridum hort and Heliconia bihai marginata, for the treatment of domestic wastewater and, through a recirculation system of the treated water, to use it for irrigation of the Phaseolus vulgaris crop.

The wetland system comprises eight horizontal subsurface flow cells in plastic boxes with a volume of 68 L (dimensions: length 41 cm, width 59 cm, and height 41 cm), filled with red tezontle rock substrate with a diameter of 8 mm and approximately 40% porosity, previously screened, planted with two different types of ornamental plants, Hippeastrum hybridum hort and Heliconia bihai marginata, in addition to conditioning polyculture and control cells; the hydraulic retention time was 5 days. This system is fed by a piping mechanism coupled to a 100 L container, which is filled weekly with domestic wastewater, sourced from sewage running 15 m from the experimental area. A 1/2″ outlet valve was adapted to the container for control of the domestic wastewater flow to the wetlands, which connects to a 1″ PVC hydraulic pipe prepared with eight connections, eight valves, and eight macro-drop solution dispensing arrangements for the supply of wastewater to each of the cells, with horizontal flow in each of them. The boxes were adapted to an outlet, also with stopcocks and PVC hydraulic piping, in addition to gooseneck faucets in each one for water collection, which are also connected to each other, thus directing the treated wastewater to each of the grow beds (Figure 2). The wetland system has two cells conditioned with Hippeastrum hybridum hort, two cells with Heliconia bihai marginata, two with polyculture, and two control cells (without planting), and from the moment it is planted, it must go through an adaptation period (one month), which is essential to ensure that the ornamental plant adapts to its new ecosystem. The wetland system was monitored for one year (January–December 2023), obtaining 12 samples of wastewater with its replicate and treated water for each of the cells.

Recirculation of treated water to the grow beds was carried out through a PVC hydraulic piping system and a stopcock connected to the drip irrigation system’s polyduct hose for a controlled supply.

• Stage 3. Experimental design of the wetland system.

Pollutant concentrations in the influent and effluent of each cell were measured monthly for 12 months, from January 2023 to December 2023. The parameters used to assess wastewater quality included chemical oxygen demand (COD), total suspended solids (TSS), total nitrogen (TN), ammonium (NH₄⁺), nitrites (NO₂⁻), total phosphorus (TP), and phosphates (PO₄³⁻). Analyses were performed according to standard laboratory methods; for example, the water temperature, pH, and electrical conductivity (EC) were also measured using the portable multiparameter Hanna model HI98121 (HANNA^® instruments, Woonsocket, RI, USA), while dissolved oxygen (DO) was measured with the Milwaukee equipment model MW600 (Milwaukee Instruments, Rocky Mount, NC, USA) [43]. An adaptation period of 2 months was established before starting the measurement of the selected parameters. After this period, pollutant concentrations were measured in the influent and effluent of each of the cells on a weekly basis for 12 months, from January 2023 to December 2023. The statistical analysis began with the verification of the data distribution, applying normality tests for establishing whether the results followed a normal distribution or not. For this purpose, the Anderson–Darling and Shapiro–Wilk tests (p < 0.05) were applied, evaluating the removal values of different pollutants—chemical oxygen demand (COD), ammonium (NH₄⁺), total nitrogen (TN), nitrites (NO₂⁻), total phosphorus (TP), phosphate (PO₄³⁻), and total suspended solids (TSS)—in each of the treatments considered. These tests were fundamental to determining the suitability of using parametric statistical methods by indicating whether the data sets conformed to a normal distribution. In total, eight experimental units were set up, distributed in three treatment units with their respective replicates. Three treatment units of plants organized in monoculture were included: cells 1 and 2 with Hippeastrum hybridum hort, cells 3 and 4 with Hippeastrum hybridum hort, cells 5 and 6 with polyculture (Heliconia bihai marginata and Hippeastrum hybridum hort), and two control cells without plants (7 and 8). Each was subjected to laboratory methods to analyze the contaminants COD, NH₄⁺, TN, NO₂⁻, TP, PO₄³⁻, and TSS.

For the cases in which the data did not meet the assumptions of normality, the Kruskal–Wallis nonparametric test was used as an alternative to compare treatments, thus avoiding possible biases derived from the use of inadequate parametric methods.

• Stage 4. Set up of two grow beds (GBs) for planting the crop and a drip irrigation system.

The area for the construction of two grow beds was assigned for monitoring the crop cycle. The measurements and conditions were the same for both; the measurements of each grow bed were 8 m long by 1.01 m wide by 1.18 m high (Figure 3). Sandy loam soil, with a pH of 6.88, was applied to grow bed 1, with pH 7.01 for number 2. Once set up, a drip irrigation system was installed.

Drip irrigation system: To optimize water distribution, it was essential to consider an efficient irrigation system, widely implemented in agriculture and based on the specific needs of the area and the crop. The selected drip irrigation system guarantees uniformity and greater irrigation coverage [44,45]. The irrigation system for the experiment was chosen by comparing efficiencies, minimum water needs, and increased production, according to the literature, also bearing in mind energy consumption and requirements (see

Table 1. Comparison of irrigation systems for agricultural crops.

Irrigation Type		Water Supply	Water Need	Water Saving %	Production Increase %	Energy Cost	Crop Type
Surface irrigation	Ridge	Irregular	Abundant	S-N	30%	High	Rice, corn, grasses, vineyards, and cotton
	Furrow	Irregular	Moderate	S-N	NR	High	Sugarcane
Pressurized irrigation	Drip irrigation	Continual	Limited	95–100%	40–85%	Moderate	Limes, lemons, tangerines, oranges, grapefruits, corn, soybeans, herbs, bananas, sugarcane, cotton, beans, potatoes, pumpkin, melons, peanuts, and chilis
	Sprinkler irrigation	Regular	Medium	80–85%	25–35%	Medium	Corn, soybeans, wheat, cotton, peanuts, potatoes, sunflowers, alfalfa, sorghum, sugar beets, and vegetables
	Micro-sprinkler irrigation	Regular	Medium–high	46–82%	27%	Medium	Nursery, frost protection, garden irrigation, horticulture, fruit growing, flowers, greenhouses, and cocoa

NR: not reported; S-N: scarce to null.

). A comparison of irrigation systems implemented in agriculture was carried out as well.

The drip irrigation system was selected based on its limited water requirement, the percentage of water savings, and its moderate energy cost. In its implementation, several components were considered for ensuring homogeneity of the conditions in both grow beds. Once the drip irrigation system was adapted to the GBs, the process of planting, growing, and harvesting the crop began.

• Stage 5. Planting, harvesting, and food safety analysis of Phaseolus vulgaris crops.

The Secretaría de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación (SAGARPA) provides that one way to improve the yield and nutritional value of this crop is to look for growing regions that can better adapt to the different environmental conditions required [46]. The study of precipitation and temperature confirmed that the area has the right conditions for the crop to grow adequately, and Phaseolus vulgaris can be an alternative for producers of the Misantla region. For the experiment, the crop was planted during the rainy season (November 23–February 2024). The considerations for planting Phaseolus vulgaris in this study are listed in

Table 2. Considerations for Phaseolus vulgaris cropping.

Item	Description
Crop	Ejotero Bean
Variety	Phaseolus vulgaris
Distance between seeds	30 cm
Seeds per vine	4
Total plant number	42
Total number of sown seeds	126
Sowing depth	4 cm
Days to germination	7 to 15 days
Vegetative development	2 to 3 weeks
Sowing depth	2–3 cm
Germination %	>91%
Blossoming	1 to 2 weeks
Pod formation	2 to 3 weeks
Seed/crop maturity	50 to 70 days

.

• Stage 6. Fourier transform infrared spectroscopy (FTIR) analysis and visual characterization of microplastics.

Microplastics are an emerging pollutant, for which the full picture is still missing. To date, no methodology or analytical methods exist for quantifying and/or characterizing them in a standardized way, nor a critical comparison or validation of them for their measurement, especially at the sampling process level. However, in most of the latest studies for their chemical identification, the Fourier transform infrared spectroscopy (FTIR) technique is applied, using a spectrophotometer equipped with a Perkin Elmer ATR module with a diamond crystal and a sampling area of 1.5 mm in an optical window of 400 to 3500 cm⁻¹ for identifying the type of polymer [47]. For this study, the pretreatment was the same for the three types of water sampled:

• Sampling: a sterile, 500 mL glass container with a metal lid was used.
• Digestion of organic matter: using potassium hydroxide and a thermo-stirrer for 24 h at a temperature of 50 °C, digestion of the organic matter in each water sample was achieved.
• Sedimentation: forty-eight hours after organic matter digestion, the samples were transferred to 60 mL glass test tubes with lids, using a glass pipette previously marked with an identification label.

Visual characterization: the methodology for the characterization of microplastics is based on the following:

• Filtration supplies: a filter holder, cellulose or glass fiber filters, fine-tipped forceps, beaker, 60 mL syringes, Petri dish, yellow isolating tape, black oil pen, optical microscope, and stereoscope.
• Filtration process: the water sample is poured into a beaker, the filter is placed in the filter holder, and the filter holder is placed in the sample container; to supply the water sample with the help of the syringe, the filter is removed with the forceps and placed in a Petri dish and marked with insulating tape.
• Drying: the drying time of the filter is 24 h.
• Visualization: the filter is visualized by means of an optical microscope and a model VE S-1, VELAB-brand stereoscopic microscope, with different lens objectives (magnifications, 2× and 4×).

3. Results

3.1. Cropping of Phaseolus vulgaris

The crop was planted according to its seasonality during a frequent rainfall period from November 2023 to February 2024.

Table 3. Phaseolus vulgaris crop data.

Phaseolus vulgaris
Seeding date	23 November 2023
Sprouting date	27 November 2023
Measurement start date	30 November 2023
Irrigation type	Drip
Requirement/Day
Irrigation	2 times a day
Harvest date	14 February 2024
Total days	84 days

describes the key dates. Grow bed soil pH measurements were acquired in the period 23 November 2023–14 February 2024, using a Hanna GroLine HI981030 Soil PH Meter. The grow bed irrigated with treated wastewater obtained a pH of 7.01, and the one irrigated with well water obtained a pH of 6.73 on average during this period. Both values are within the range indicated by SAGARPA [40] in its national agricultural plan for 2017 to 2030. This demonstrates that the adequate pH fluctuates between 6.6 and 7.5, as within these limits, and most of the soil nutrients express their maximum availability. Basing on the Misantla area’s study and description, the analysis of isotherms and isohyets reveals that the experimental area has an average annual rainfall of 2200 to 2280 mm and a temperature range of 22 °C, which are ideal conditions for normal crop growth.

Concerning the plants’ biometric development, the study included the keeping of a log of plant height growth from germination onwards, measured every fifteen days, in cm, using a metric ruler as a tool. The average growth data of the crop in the grow bed irrigated with wetland-treated wastewater was 26 cm, and for the bed irrigated with well water, it reached an average of 21 cm. Also, for both cases, it was noted that the greatest height of the plants occurred in the period of 45 to 60 days, after which the plants began to decay. The moment of harvesting occurred 15 days after the maturity of the pod, after which the harvesting work began, which consisted of pulling up each plant completely and subjecting it to a drying time of 10 days under a roof, hanging from a wire, and marking each plant with its corresponding number, without involving any plastic material.

For crop yield, after drying the fruit, the total product was weighed and de-sheathed. For this process, a BLVEI, 1–10 kg capacity, high-precision digital weight scale was used. The area where the weighing and packaging activities were carried out was on a flat wooden base, and latex gloves were used following sterilization recommendations. The beans collected from the pods were placed in plastic Ziplock bags to be sent to the laboratory for safety analysis. The total weight of the product from the grow bed fed with water treated by constructed wetlands was 788 g, while the grow bed irrigated with well water yielded 720 g of product. It is worth mentioning that the number of pods in the plants was higher for those irrigated with treated water, as 895 pods were counted for the 21 plants, while the plants irrigated with well water produced a total of 652 pods. The differences between crops could be attributed to the properties of the wastewater treated from the wetland system.

For the food safety analysis of Phaseolus vulgaris crops, based on biological agents or pathogens that may be present in the product, the GisenaLabs, Grupo Integral de Servicios Fitosanitarios, ENA S.A. de C.V. laboratory was requested to analyze the presence of Escherichia coli and Salmonella in the Phaseolus vulgaris variety crop. The analysis conducted on the Phaseolus vulgaris crop yielded negative results for Escherichia coli and Salmonella.

3.2. The Wetland System

During the one-year study period (January–December 2023), the average temperature was 26 °C, the relative humidity was 87%, and the average solar radiation was 155 W/m². Hippeastrum hybridum hort grew to 35 cm in height, and Heliconia bihai marginata grew to 90 cm in height. The average light intensity of this study was 1235.24 lux, which is within the intensity ranges for tropical zones (500 to 2100 lux) [20]. The hydraulic retention time was 5 days.

Table 4. Pollutant removal by the constructed wetland system.

		Hippeastrum hybridum Hort	Heliconia bihai marginata	Polyculture	Control
COD (mg/L)	IC	908.17 ± 17.45
	EC	105.10 ± 1.64	88.4 ± 0.190	10.59 ± 0.35	536 ± 9.10
	% of removal	88.4 ± 0.190	93.64 ± 0.161	98.83 ± 0.04	40.77 ± 1.16
NH4++ (mg/L)	IC	35.91 ± 1.30
	EC	7.47 ± 0.33	3.36 ± 0.11	0.40 ± 0.05	23.40 ± 1.25
	% of removal	78.99 ± 1.012	90.54 ± 0.334	98.89 ± 0.13	33.01 ± 4.50
TN (mg/L)	IC	87.56 ± 3.60
	EC	14.72 ± 0.42	8.71 ± 0.18	3.38 ± 0.10	50 ± 1.56
	% of removal	82.80 ± 0.736	89.85 ± 0.363	96.05 ± 0.18	42.06 ± 2.14
NO2−− (mg/L)	IC	184.74 ± 4.77
	EC	2.97 ± 0.13	11.30 ± 0.32	1.77 ± 0.05	107 ± 2.41
	% of removal	88.94 ± 0.377	93.85 ± 0.183	99.03 ± 0.04	41.24 ± 1.96
TP (mg/L)	IC	20.64 ± 0.76
	EC	2.97 ± 0.13	1.43 ± 0.04	0.19 ± 0.01	12 ± 0.49
	% of removal	85.53 ± 0.709	93.04 ± 0.218	99.05 ± 0.04	41.12 ± 2.71
PO43−− (mg/L)	IC	41.00 ± 0.88
	EC	5.63 ± 0.19	2.71 ± 0.08	0.40 ± 0.0038	23 ± 1.53
	% of removal	86.23 ± 0.480	93.36 ± 0.213	99.03 ± 0.02	43.66 ± 3.83
TSS (mg/L)	IC	111.36 ± 4.56
	EC	21.94 ± 0.46	13.29 ± 0.22	5.67 ± 0.10	59 ± 1.65
	% of removal	80.21 ± 0.435	87.97 ± 0.300	94.84 ± 0.16	46.41 ± 1.65

IC: input concentration; EC: exit concentration.

below describes the results of the annual mean concentrations.

Statistically significant differences were observed in the removal efficiency between the systems planted with Hippeastrum hybridum, Heliconia bihai margarita, the polyculture, and the control. All analyses were performed at a significance level of 5% (p ≤ 0.05).

Influent domestic wastewater includes high percentages of pollutant load (COD, NH₄⁺, TN, NO₂⁻, TP, PO₄³⁻, and TSS). The cells with Hippeastrum hybridum hort had a removal efficiency of 88.40%; the cells with Heliconia bihai marginata had a removal efficiency of 93.64%; and the polyculture had a removal efficiency of 98.83% for COD. The efficiency of the control cell was lower, with an annual average of 40.77%. This demonstrates the importance of ornamental plants for these systems and their adaptation to a tropical climate. The removal rates for the other pollutants followed a similar pattern. The domestic wastewater of the experiment contained ammonium (NH₄⁺) at 35.91 mg/L. Cells conditioned with Hippeastrum hybridum hort achieved a removal of 78.99% for this contaminant, while those with Heliconia bihai marginata reached 90.54%. The polyculture reached 98.89% and the control only 33.01%. For TSS with an influent load of 111.36 mg/L, polyculture cells achieved an average removal efficiency of 94.84 over a one-year period; cells with Hippeastrum hybridum hort, 80.21%; Heliconia bihai marginata, 87.97%; and control, 46.41%. This design has been considered in cells with ornamental plants, with polyculture, based on its mention in the literature [21,25]. The cell with Hippeastrum hybridum hort removed contaminants at an average rate of 84%; the cell with Heliconia bihai marginata, 98%; the cell with polyculture, 98%; and the control cell, 41%. For the pollutants COD, NH₄⁺, TN, NO₂⁻, TP, PO₄³⁻, and TSS, the removal rate was found to be higher than 90% for both polyculture and Heliconia bihai marginata.

3.3. Fourier Transform Infrared Spectroscopy (FTIR) Analysis

Once the samples were obtained, they were sent off for analysis using the PerkinElmer UATR Two FTIR system with an ATR module. For each type of water analyzed (a = domestic wastewater, b = domestic wastewater treated by constructed wetlands, and c = well water), the results are shown in graphs (Figure 4) for groups of three samples, respectively. The ranges were interpreted based on the literature findings [48,49].

Table 5. Ranges found in samples of three types of water, FTIR analysis.

(a) Domestic Wastewaters
Sample No.	Range	Polymer
M1	1633	PU, EVA, PA, PET, and PMMA
	1400–1480	PP, PS, PET, and PA
M2	1634	PU, EVA, PA, PET, and PMMA
	1044	PTFE
M3	1634	PU, EVA, PA, PET, and PMMA
(b) Constructed Wetland-treated Domestic Wastewaters
Sample No.	Range	Polymer
M1	1633	PU, EVA, PA, PET, and PMMA
M2	1633	PU, EVA, PA, PET, and PMMA
M3	1633	PU, EVA, PA, PET, and PMMA
(c) Well Water
Sample No.	Range	Polymer
M1	1634	PU, EVA, PA, PET, and PMMA
	1044	PP, PS, PET, and PA
M2	1634	EVA and Aliphatic polyester
	1044	PTFE
M3	1634	EVA and Aliphatic polyester
	1044	PTFE

describes the prominent peaks at the reported polymer ranges. Although only three samples per point were analyzed (a: M1, M2, and M3; b: M1, M2, and M3; c: M1, M2, and M3), the FTIR analysis allowed for the identification of polymers in all nine samples.

However, other studies recommend the use of RAMAN for the determination of the total number, structure, and size of particles, highlighting their complementary functions [47]. Ranges were found in samples of three types of water analyzed via FTIR; the polymers found were Polyurethane (PU), Ethylene Vinyl Acetate (EVA), Polyamide (PA), Polyethylene Terephthalate (PET), Polymethyl Methacrylate (PMMA), Polypropylene (PP), Polystyrene (PS), Polytetrafluoroethylene (PTFE), Aliphatic polyester (APP).

A correlation might exist between the MPs present in the analyzed samples and the usual activities at the place where the wastewater is discharged. Although the experimental design did not include a standardized protocol for quantifying these particles in the influent and effluent of the constructed wetland system, the adopted approach allowed for their identification through FTIR spectroscopy and visual observation. This represents a valuable first step toward understanding the behavior of these contaminants in nature-based treatment systems.

Despite the methodological limitation regarding direct quantification, the results provide evidence of the wetland system’s ability to intercept plastic particles in a decentralized treatment context. Future research should focus on more precise monitoring methods, such as the use of synthetic wastewater with controlled concentrations of microplastics, which would enable more detailed performance evaluations. Furthermore, this would facilitate the analysis of the role played by different components of the wetland system, such as the substrate type, vegetation, and hydraulic configuration, in the retention and removal processes of these emerging pollutants.

3.4. Visual Characterization of Microplastics

As per the classification by Crawford et al. [49] and methodology for visual characterization of MPs, we classified our findings as described in

Table 6. Classification of microplastics.

Sample	Amount	Color	Shape
Domestic wastewater	1	Purple	Fiber
Domestic wastewater	4	Pale blue	Fragment
Domestic wastewater	10	Deep blue	Fiber
Domestic wastewater	1	Transparent	Fragment
Well water	2	Red	Fiber
Well water	1	Pale blue	Fiber
Well water	1	Deep blue	Fiber
Well water	5	Transparent	Fiber
Well water	2	White	Fragment
Cell 8	1	Pale blue	Fragment
Cell 8	2	Red	Fragment
Cell 7	2	Deep blue	Fragment
Cell 6	0	NF	NF
Cell 5	0	NF	NF
Cell 4	0	NF	NF
Cell 3	1	Deep blue	Fiber
Cell 2	0	NF	NF
Cell 1	1	Deep blue	Fiber

NF: not found.

below. The MPs found were sorted by color and shape. The total microplastic particles in the form of fiber reached a concentration of 11 particles per half a liter of water (500 mL) and that of microplastic particles in the form of fragments was 6 per half a liter of water (500 mL).

The pictures in Figure 5 are of the filters through which the treated water samples from each of the cells of the constructed wetlands were run, both from the untreated domestic wastewater and the well water.

Microplastic fibers were observed in the filters of cells 1 and 3, highlighted here in a red circle. However, only one fiber strand was shown on each filter (blue fiber), and the two pictures of cells 7 and 8 show the highest number of microplastic fibers: red, deep blue, and light blue.

The highest concentration of microplastics found was in the untreated domestic wastewater filters, with fibers and fragments of deep blue, pale blue, purple, and transparent colors.

In the well-water filters, microplastic fibers and fragments were also identified, mostly of transparent and red color, but also white, pale blue, and deep blue.

4. Discussion

CWs have proved to be effective systems for removing pollutants from wastewater from different sources. In our experiment, Hippeastrum hybridum hort and Heliconia bihai marginata removed pollutants such as COD, NH₄⁺, TN, NO₂⁻, TP, PO₄³⁻, and TSS with an efficiency of 84% and 98%, respectively, on average. This is similar to the results of Madera et al. [41], who used Heliconia in constructed wetlands for the phytoremediation of leachate from landfills and sewage, obtaining a removal efficiency of 66% and 72% for COD and NH₄⁺ respectively. Similarly, Viveros et al. [32], in their work on constructed wetlands for the treatment of municipal and pig farming wastewater using Heliconia, reported that this plant furthered the removal of pollutants with an efficiency of 93.42%. However, they have been studied little in terms of microplastic removal potential. The study by Shukla et al. [42], in which planted horizontal subsurface flow CWs and a system with floating plants as secondary treatment after WWTPs for domestic wastewater treatment, reports high efficiencies for the removal of MPs: 97.42% for subsurface flow wetlands and 98.13% for the floating plant wetland system. Following the same idea of using CWs for the removal of MPs, a study was carried out on free-flow CWs fed with a secondary stream of treated wastewater to a sedimentation pond with shallow vegetation, reporting that it retained between 37.8 and 92% of the MPs [42]. However, it was also reported that treatment wetlands of this type may release MPs during high precipitation events. Different wetland designs are known. For example, Cabrera et al. [23] conducted a study of laboratory-scale vertical wetlands filled with sand as an unplanted filtering medium, taking into account the influence of biofilm between the filter medium and earthworms for the removal of MPs. After 45 days, they reported that the MPs were retained in the first 10 cm, with a 99% removal efficiency. They also mentioned that a larger grain size and the presence of worms have little effect on the removal efficiency. Chen et al. [27] conducted investigations using CWs and MPs in various shapes (films, fragments, and fibers) and sizes (0.5 mm and 2–4 mm). Their findings revealed an MP removal rate of 81.63% in surface flow wetlands and 100% in horizontal subsurface flow wetlands. In addition, they noted that filtration is very important for retaining the MPs, but that they could pass through the openings between the substrate if their shape was just right.

When properly conditioned with different plant species, substrates, and flow configurations, CWs demonstrate the ability and efficiency for retaining and/or removing particles known as MPs [50].

Samples analyzed via Fourier transform infrared spectroscopy (FTIR) showed that this type of domestic wastewater treatment can remove or retain the following types of polymers: PP, PS, PT, and PTFE. However, these treated water samples still contained polymers of PU, EVA, PA, PET, and PMMA, suggesting that these polymers move through the sewage flow between the substrate and the roots of ornamental plants. This transport is likely to be influenced by the particle size, and the type of horizontal subsurface flow in the wetland system may also play a role.

5. Conclusions

The results of this study confirm the presence of microplastics in well water, untreated domestic wastewater, and in the treated effluent from the constructed wetland system, as identified through FTIR spectroscopy and visual characterization. In the treated water samples, a total of 11 fibers and 6 microplastic fragments were detected per 500 mL of water. Although removal efficiency was not quantified, these findings suggest that horizontal subsurface flow constructed wetlands with ornamental plants have the potential to retain MPs in decentralized wastewater treatment systems.

High removal efficiencies were also observed for conventional pollutants such as COD, NH₄⁺, TN, NO₂⁻, TP, PO₄³⁻, and TSS. The species Hippeastrum hybridum hort and Heliconia bihai marginata achieved average removal rates ranging from 84% to 98%, contributing significantly to the overall performance of the system.

In order to reuse the treated water for the irrigation of agricultural crops and to assess its potential effects on plant development, full implementation of the recirculation system to the cultivation beds was achieved. The sowing, growth, maturation, and harvesting stages of Phaseolus vulgaris were monitored, although only through visual records, including observed biometric development of the crop. The potential impact of microplastics on plant biomass and physiological processes remains a subject for future investigation.

This research contributes to the understanding of microplastic behavior in nature-based sanitation technologies and provides a methodological foundation for future studies, incorporating synthetic wastewater with known microplastics concentrations and controlled sampling protocols to enable accurate quantification and risk assessment in agricultural reuse applications.