The Influence of Sewage on the Quantitative and Functional Diversity of Nematode Communities in Constructed Wetlands (VFCW): Analysis of Trophic Relationships Using Canonical Methods

Abstract

Given the increasing demand for water and the need to reduce energy consumption, modern wastewater treatment systems should be characterised by high pollutant removal efficiency while consuming low resources. Hydrophytic wastewater treatment plants with vertical flow through a soil-plant bed (VFCW) are one solution that meets these requirements. The efficiency of these systems largely depends on the biological activity of the bed, of which free-living soil nematodes are an important component. The study presented in this paper aimed to assess the relationship between the quality of domestic wastewater flowing into VFCW beds and the abundance and trophic structure of soil nematode communities. The analysis was carried out on two real-world sites, where VFCW beds were the third stage of the plant bed system. Both treatment plants received only domestic wastewater. Statistical analysis showed no significant differences (p > 0.05) in the physicochemical composition of the wastewater flowing into the two treatment plants, indicating homogeneous system feed conditions. Nevertheless, canonical correspondence analysis (CCA) showed that the relationships between effluent parameters and the abundance of individual nematode trophic groups differed in each bed, suggesting the influence of local environmental and biocenotic conditions. In particular, bacterivorous nematodes—key to bed function—were shown to be sensitive to different sets of variables at the two sites despite similar effluent composition. These results confirm that the rhizosphere—a zone of intense interactions between plant roots, microorganisms, and soil microfauna—plays a critical role in shaping the biological activity of the bed. Nematodes, particularly bacterivorous nematodes, support the mineralisation of organic matter and nutrient cycling, resulting in increased efficiency of treatment processes. The stability of the total nematode abundance, irrespective of inflow conditions, demonstrates the bed biocenosis high ecological resilience to external disturbances. The study’s results highlight the importance of an ecosystem approach in designing and managing nature-based solutions (NBS) treatment plants, which can be a sustainable component of sustainable water and wastewater management.

1. Introduction

With increasing demographic and urbanisation pressures and intensification of economic activities, the world is currently facing a surge in demand for freshwater—both for domestic and industrial/agricultural purposes [1,2]. At the same time, there are growing difficulties in securing access to water of adequate quality in many regions of the world, especially in the context of global climate change and degradation of aquatic ecosystems [3]. Therefore, measures to effectively rationalise water use and increase the share of water reuse are becoming key elements of sustainable development strategies [4].

According to projections by Christou et al. [5], global water abstraction is currently around 359.4 × 10⁹ m³ per year and is expected to increase by 50% by 2050, mainly due to the intensification of food and industrial production. Only about 20% of the world’s wastewater is effectively treated—depending on the region, this figure ranges from 8% in developing countries to 70% in highly industrialised countries [5]. Growing environmental awareness, the need to reduce greenhouse gas emissions, and increase the energy self-sufficiency of municipal systems mean that wastewater treatment systems are currently undergoing a dynamic transformation towards a circular economy (CE) model [6].

These changes are formally reflected in a new EU directive [7], which makes it mandatory to effectively remove biogenic pollutants and micropollutants, recover water and secondary raw materials, and implement solutions that reduce greenhouse gas emissions and energy consumption.

In this context, the importance of nature-based solutions (NBS) technologies is growing, mainly constructed wetlands (CW), and in particular Vertical Flow Constructed Wetlands (VFCW) systems, which allow effective wastewater treatment with low energy requirements, high biological efficiency, and the potential for water resource recovery [8].

Nature-based solutions such as VFCW systems address water scarcity while meeting water treatment needs. This is due to the construction (design) and operating principle of VFCW systems. The components of VFCW systems include a soil-plant bed and a denitrification pond (the last element of the wastewater treatment plant). Treated sewage can overflow the slopes of the denitrification pond and enter the ground and then into the groundwater. They supply local groundwater. In addition, rainwater is retained in the denitrification pond, which improves the local climate.

Hydrophytic wastewater treatment plants enable the simultaneous delivery of multiple ecosystem services: nutrient load reduction, heavy metal fixation, degradation of organic micropollutants, and protection of microbial biodiversity and soil microfauna [9]. The critical component determining the effectiveness of these systems is the soil-plant bed, whose structure and biological activity determine the effectiveness of self-purification processes [10]. As shown in the study by Bagińska et al. [11], soil biocenosis plays a key role in regulating organic matter transformation processes and biogeochemical cycles. A fascinating group of organisms is nematodes (Nematoda), which, depending on the trophic group represented, have diverse ecological functions, ranging from influencing the rate of organic matter mineralisation to controlling the microbial population in the soil [12]. However, there is still a lack of systematic studies on the influence of physicochemical parameters of wastewater (such as organic load, nutrient concentration, pH, and heavy metal content) on the trophic and functional structure of nematode assemblages in VFCW systems. While several studies have examined nematode communities in agricultural soils and some have investigated nematodes in constructed wetlands, systematic investigations using multivariate statistical approaches (particularly CCA) to link specific wastewater physicochemical parameters with nematode trophic group responses in VFCW systems remain limited.

Atira and Kakouli-Duarte [13] investigated the effect of soil fertilisation with organic and mineral fertilisers on the abundance and trophic groups of soil nematodes. The research results showed that bacterivorous nematodes are the most resistant to changing soil conditions and develop even with high fertiliser concentrations in the soil. Organic fertilisers are more friendly to the soil biocenosis than inorganic fertilisers. Recycling-derived fertilisers (RDFs) have been shown to quickly deliver nutrients to the soil and are an alternative to mineral fertilisers. However, their impact on the abundance and trophic groups of nematodes requires further research.

The latest publications concern the effectiveness of antibiotic (oxytetracycline) removal in soil-plant deposits [14], but no one has yet examined the impact of sewage quality on soil nematodes inhabiting these deposits.

While nematodes are recognised as important soil fauna in terrestrial ecosystems and their ecological roles have been studied in agricultural contexts, systematic investigations of how wastewater physicochemical parameters specifically influence nematode functional (trophic) diversity in VFCW systems remain limited. Particularly lacking are studies that: (1) use multivariate statistical approaches to identify which wastewater parameters drive abundance patterns of specific trophic groups, (2) examine these relationships in real-world, small-scale domestic systems, and (3) compare patterns across sites to distinguish general versus site-specific responses. Our study addresses these gaps by applying CCA to link 16 wastewater parameters with five nematode trophic groups across two operational VFCW systems.

Analysis of these relationships using multivariate statistical methods, including canonical methods (e.g., CCA—canonical correspondence analysis), can provide valuable information on the ecological mechanisms in hydrophytic beds and allow optimisation of their design and management in the context of treatment efficiency. Therefore, this paper aims to investigate the influence of wastewater characteristics on the quantitative and functional diversity of nematode assemblages in hydrophytic wastewater treatment plants (VFCW) and identify key trophic relationships using canonical methods. The results will contribute to the knowledge of the biological basis of nature-based solutions (NBS) systems and enable their further development to align with modern water, wastewater, and climate policy requirements.

2. Materials and Methods

2.1. Study Objects

The objects of the study were two soil-plant beds that were the third component of Vertical Flow Constructed Wetlands (VFCW), operating as part of a proprietary plant bed system (Figure 1). Soil-plant deposit number 1 was located in the Vertical Flow Constructed Wetland (VFCW) on the individual farm Jabłonowo-Wypychy 10 (Sokoły commune, Podlaskie Voivodeship). Soil-plant deposit number 2 was located in the Vertical Flow Constructed Wetland (VFCW) on the individual farm Rzące 25 (Sokoły commune, Podlaskie Voivodeship). These systems were located on private individual farms and were used to treat domestic wastewater from two households. Both treatment plants operated under real-world conditions, such as low-flow, backyard hydrophytic systems serving individual households.

The principle of operation of the VFCW system is that domestic sewage first flows by gravity to the first device—the septic tank (1). In the septic tank (2) the processes of sedimentation and flotation take place. Then, the supernatant liquid flows by gravity to the second device—the pumping station (3). After preliminary treatment, the wastewater is pumped through a pipeline (4) to the soil-plant bed (5), which is composed of various filter layers. In the filter bed, as a result of physical, chemical and biological processes, vertically flowing wastewater is purified. At the very bottom of the deposit there is a drainage (10). Above this layer there is a layer of medium sand (8) and above that an organic layer (7). After percolating through the bed, the sewage flows by gravity through an intermediate well (11) to the denitrification pond (12), where the processes of denitrification and sewage purification take place with the participation of plants (14) and aquatic organisms.

The plant-based wastewater treatment plants in which the research object operated formed a decentralised wastewater treatment system in rural areas.

2.2. Sampling Points and Method

Sewage samples for physicochemical analysis and soil samples for nematode abundance and trophic group analysis were collected each season: spring, summer, and autumn. Three samples were collected from the Jabłonowo-Wypychy 10 VFCW and five samples were collected from the Rzące 25 VFCW. The measurement period was 1.5 years.

Wastewater samples were taken from the pumping station (3) and from the intermediate well (11). The following parameters were examined in the wastewater samples: pH, temperature (Temp), dissolved oxygen (DO), alkalinity (Alk), biochemical oxygen demand (BOD₅), chemical oxygen demand (COD), total suspended solids (TSS), total phosphorus (TP), phosphate phosphorus (P-PO₄), phosphorus pentoxide (P₂O₅), total nitrogen (TN), ammonium nitrogen (N-NH₄), nitrate nitrogen (N-NO₃), nitrite nitrogen (N-NO₂), chlorides (Cl), iron (Fe). Wastewater samples were taken in accordance with PN-ISO 5667-10:2020 [15]. The collected samples were protected from damage, spillage, and contamination during transport. The sample vessels were placed in a tourist fridge with cooling inserts to prevent the temperature of the collected samples from rising. The wastewater samples were transported to the laboratories in the shortest possible time. Subsequent determinations of wastewater parameters, i.e., biochemical oxygen demand (BOD₅), chemical oxygen demand (COD), total suspended solids (TSS), total nitrogen (TN) and its compounds: ammonium nitrogen (N-NH₄), nitrate nitrogen (N-NO₃), nitrite nitrogen (N-NO₂), total phosphorus (TP) and its compounds (phosphate phosphorus (P-PO₄), phosphorus pentoxide (P₂O₅)), chlorides (Cl), and iron (Fe), were performed under laboratory conditions. Biochemical oxygen demand (BOD₅) analysis was performed according to ISO 5815-1:2019 [16]. Wastewater samples were not filtered before the BOD₅ measurement. The manometric method was used to determine this indicator, using the OxiTop kit and a thermostatic cabinet (hothouse). The weighing method determined total suspended solids, following the standard EN 872:1996 [17]. Determination of total suspended solids content consisted of draining a defined volume of wastewater sample through a tissue filter, then drying at 103–105 °C for at least one hour to a constant weight of a clean filter. Measurements of wastewater parameter concentrations, i.e., chemical oxygen demand (COD), total nitrogen (TN) and its compounds: ammonium nitrogen (N-NH₄), nitrate nitrogen (N-NO₃), nitrite nitrogen (N-NO₂), total phosphorus (TP) and its compounds (phosphate phosphorus (P-PO₄), phosphorus pentoxide (P₂O₅)), chloride (Cl), iron (Fe) were performed using appropriate Merck reagents and a spectrophotometer Orion AquaMate 8000 UV-VIS. We calibrated each new batch of reagents to account for differences in reagent composition between batches and other factors affecting the accuracy of the fixed-curve method. Each time, we prepared a blank sample and a sample with a known concentration within the method’s range so that the instrument could calculate a correction factor. This correction factor was then applied to the current test method. Three replicate readings of the concentration of each contaminant in the sample were performed.

Table 1. List of standards provided by reagent manufacturer for compounds tested and error margins [own elaboration].

Name of Association	Type of Standard
NH4+	EPA Method 350.1 [18]; ISO 7150-1:1984 [19]; DIN 38406-5:1983-10 [20] Error margins: ±1.0 mg [mgN-NH4·dm−3]
NO2−	EPA Method 354.1 [21]; APHA-4500 NO2− B [22]; DIN EN 26777 [23] Error margins: ±0.0028 mg [mgN-NO2·dm−3]
NO3−	DIN 38405-9 [24] Error margins: ±0.13 mg [mgN-NO3·dm−3]
TN	ISO 11905-1:1997 [25]; DIN 38405-9 [24] Error margins: ±1.1 mg [mgN·dm−3]
TP	EPA Method 365.3 [26]; APHA Method 4500-P [27]; ISO 6878:2004 [28] Error margins: ±0.09 mg [mgP·dm−3]
Cl−	APHA Method 4500-Cl [29]; EPA-NERL: 325.1 [30] Error margins: ±2.8 mg [mgCl·dm−3]
Fe	ISO 8466-1 [31]; DIN 38402 A51 [32] Error margins: ±0.016 mg [mgFe·dm−3]

shows the standards for each test procedure and the standard deviation of the method (margins of error).

In order to obtain the values of specific physico-chemical pollutants in wastewater flowing through soil-plant beds (5), the average value of the concentration of pollutants was determined between the wastewater intake point: pumping station (3) and the wastewater intake point: intermediate well (11). Soil samples for nematode abundance and trophic groups determination were taken from the top layer of the soil-plant beds (5).

Wastewater samples were collected at the inlet to the treatment plant’s soil-plant bed and at the outlet from the soil-plant bed, while nematode samples were obtained directly from the biological bed. We decided that averaging the values of physicochemical parameters from both measurement points would allow us to approximately reflect the environmental conditions prevailing in the deposit, i.e., where the organisms were collected. This approach seemed justified because the deposit is located between these two points and the conditions within it are an indirect effect of changes occurring between the input and output.

Soil samples were taken using a method commonly used in ecological surveys, which involves cutting soil cylinders with an Egner stick with a diameter of 1.6 cm and a cross-sectional area of 2 cm² to a depth of 20 cm. Soil samples were placed in sealed bags made of polyethylene film and transported under refrigeration to the research laboratory. A modified Baermann method was used to extract (dislodge) nematodes from the soil [33]. This method involves the active passage of nematodes from the sample to water through a porous tissue filter and a filter gauze with a mesh diameter of 100 μm. A modification of Baermann’s original method involved using plastic holders where strainers were placed. This technique is used for the extraction of live nematodes [34]. The extraction process lasted for 24 h. The resulting nematode suspension was incubated in a water bath at to 60 °C for 2 min. Formalin (aqueous formaldehyde solution) was then added at a concentration of 36÷38% to preserve the nematodes so that the final concentration was approximately 4% [35]. A 5 mL subsample of the solution was taken from each sample for direct counting. The result was then converted to the entire sample volume. A stereoscopic microscope was used for nematode counting. The results of the nematode counts were converted to 1 m² concerning an Egner stick area of 2 cm². Five trophic groups of nematodes were identified: bacterivorous, fungivorous, herbivorous, predatory, and omnivorous [36]. The affiliation of nematodes to individual trophic groups was assessed based on morphological characteristics using a light microscope. Bongers’ keys were used to determine nematodes [37]. Specifically, identification was based on morphological features: (1) buccal cavity morphology (2) oesophageal structure, and (3) overall body morphology including shape and size. Bacterivorous nematodes typically lack stylets and possess simple tubular stomas. Fungivorous nematodes are characterised by short stylets adapted for fungal feeding. Herbivorous nematodes have longer, hollow stylets designed for piercing plant cells and extracting cellular contents. Predatory nematodes exhibit large buccal cavities equipped with teeth. Omnivorous nematodes display intermediate morphological characteristics, reflecting their diverse feeding strategies. These morphological criteria allowed for reliable assignment of individual nematodes to their respective trophic groups under Olympus BX-50 (Tokio, Japan) light microscope at 200–400 magnification.

2.3. Performing a Canonical Correspondence Analysis (CCA)

Canonical correspondence analysis (CCA) is one of the multivariate analyses of ecological data [36,38]. A canonical correspondence analysis (CCA) was carried out separately for the two soil-plant deposits functioning as the third component of the Vertical Flow Constructed Wetlands (VFCW) on the individual farms Jabłonowo-Wypychy 10 and Rzące 25. Canonical correspondence analysis (CCA) was used to investigate the relationship between nematodes in soil-plant deposits and the quality of wastewater flowing through these deposits.

The assumptions for CCA are (1) the unimodal nature of the variables (bell-shaped environmental gradient) and (2) the lack of strong collinearity between environmental variables. The unimodal nature of the variables was verified using DCA analysis. Despite the linear nature of the variables, CCA analysis was chosen due to the ecological nature of the variables and the ease of interpreting the relationships between vectors of environmental variables. Strongly collinear variables (BOD, DO, P-PO₄, P₂O₅, Alk in Figure 2 and P-PO₄, P₂O₅ in Figure 3) were left in the analysis due to the relatively small number of environmental variables that are supposed to explain the abundance of trophic groups of nematodes.

The following data were used to perform canonical correspondence analysis (CCA):

-

Physicochemical parameters in the wastewater flowing through the beds, calculated as the average value between the sampling point, pumping station (3), and the wastewater collection point, intermediate well (11): pH, temperature (Temp), dissolved oxygen (DO), alkalinity (Alk), biochemical oxygen demand (BOD₅), chemical oxygen demand (COD), total suspended solids (TSS), phosphate phosphorus (P-PO₄), phosphorus pentoxide (P₂O₅), total phosphorus (TP), total nitrogen (TN), ammonium nitrogen (N-NH₄), nitrate nitrogen (N-NO₃), nitrite nitrogen (N-NO₂), chlorides (Cl), iron (Fe);

-

Abundance and trophic groups of nematodes inhabiting the deposits.

2.4. Performing a Mann–Whitney–Wilcoxon Test

The Mann–Whitney–Wilcoxon test was used to investigate the occurrence of statistically significant differences between physicochemical parameters in wastewater flowing to soil-plant beds in the Jabłonowo-Wypychy 10 wastewater treatment plant and in the Rzące 25 wastewater treatment plant. The Mann–Whitney–Wilcoxon test, also known as the Mann–Whitney U test, is one of the most popular nonparametric tests for verifying the significance of differences between the medians of a study variable, most often in two populations [39,40,41]. The most significant parameter of the test is the “p-value” parameter. If the value of the “p-value” parameter is above 0.05 (p-value > 0.05), then there is no statistically significant difference between the two study groups; if it is below 0.05 (p-value < 0.05), then there is a statistically significant difference.

In the case of the Mann–Whitney–Wilcoxon U test, the Shapiro–Wilk test was used to assess the normality of the distribution of variables. It was found that some variables had a distribution close to normal, while some variables showed deviations from normality. Due to this duality of data, it was decided to use the nonparametric Mann–Whitney–Wilcoxon U test, which can also be applied to variables with a distribution close to normal. It should be noted, however, that this test has slightly lower statistical power than the standard Student’s t-test for normally distributed data.

All statistical analyses were performed in the R environment programmes (version 4.2.2), including R Commander and Statistica 13.0, PAST 4.0.

3. Results and Discussion

3.1. Physicochemical Parameters of Wastewater Flowing Through the Soil-Plant Bed and the Abundance and Trophic Groups of Nematodes Inhabiting This Bed in Jabłonowo-Wypychy 10

The results of physicochemical studies of wastewater flowing through the filter bed in the Jabłonowo-Wypychy 10 wastewater treatment plant, as well as the number and trophic groups in this deposit, are presented in

Table 2. Concentration values of physicochemical parameters in sewage flowing through soil-plant bed in Jabłonowo-Wypychy 10.

St.					Av.	SD	CV [%]
pH	[-]	7.1	7.2	7.2	7.2	0	0
Temp	[°C]	18.6	19.7	20.1	19.5	1	5
DO	[mgO2·dm−3]	0.3	2.5	1.1	1.3	1	77
Alk	[mmol·dm−3]	9.5	12.0	8.6	10.0	2	20
BOD	[mgO2·dm−3]	25.5	150.5	77.0	84.3	63	75
COD	[mg·dm−3]	399.6	303.4	211.7	304.9	94	31
TSS	[mg·dm−3]	14.0	120.0	3031.0	1055.0	1712	162
P-PO4	[mgP-PO4·dm−3]	6.2	23.1	0.5	9.9	12	121
P2O5	[mgP2O5·dm−3]	14.2	53.1	1.0	22.8	27	118
TP	[mgP·dm−3]	20.3	25.3	12.5	19.4	6	31
TN	[mgN·dm−3]	138.3	145.8	111.7	131.9	18	14
N-NH4	[mgN-NH4·dm−3]	67.1	132.8	6.6	68.8	63	92
N-NO2	[mgN-NO2·dm−3]	1.5	0.5	2.1	1.4	1	71
Cl	[mgCl·dm−3]	227.4	240.9	129.9	199.4	61	31
Fe	[mgFe·dm−3]	0.1	0.1	0.1	0.1	0	0
N-NO3	[mgN-NO3·dm−3]	12.2	7.6	26.7	15.5	10	64

Notes: St.—statistical analysis, Av.—average, SD—standard deviation, CV—coefficient of variation.

and

Table 3. Abundance and trophic groups of nematodes in soil-plant bed in Jabłonowo-Wypychy 10.

St.	Nematode Abundance	Bacterivor.	Pred.	Fungivor.	Herbivor.	Omnivor.
	[N·m−2]
	567,000.0	294,840.0	0.0	215,460.0	28,350.0	28,350.0
	1,466,000.0	1,143,480.0	0.0	249,220.0	73,300.0	0.0
	1,161,000.0	777,870.0	0.0	255,420.0	69,660.0	58,050.0
Av.	1,064,666.7	738,730.0	0.0	240,033.3	57,103.3	28,800.0
SD	457,176	425,672	0	21,506	24,968	29,028

Notes: Bacterivor.—bacterivorous, Pred.—predatory, Fungivor.—fungivorous, Herbivor.—herbivorous, Omnivor.—omnivorous.

. The pH in the wastewater flowing through the soil-plant bed was neutral. The wastewater temperature (Temp) was quite high, ranging from 18.6 to 20.1 degrees Celsius. The dissolved oxygen (DO) content in the wastewater flowing through the bed was from 0.3 to 2.5 mgO₂·dm⁻³. The average value was 1.3 mgO₂·dm³. The alkalinity of the wastewater ranged from 8.6 to 12.0 mmol·dm⁻³. The average value was 10 mmol·dm⁻³. The biochemical oxygen demand (BOD) in wastewater flowing through the filter bed ranged from 25.5 to 150.5 mgO₂·dm⁻³. It should be noted that the wastewater entering the bed is already pre-treated in the septic tank (site number 2 in Figure 1). A very significant biochemical oxygen demand (BOD) reduction of approximately 90% occurs in the septic tank. Chemical oxygen demand (COD) ranged from 211.7 to 399.6 mgO₂·dm ⁻³. The average value was 304.9 mgO₂·dm⁻³.

The concentration of total suspended solids (TSS) in the wastewater flowing through the bed was in a wide range from 14 to 3031.0 mg·dm⁻³. The average value was very high and amounted to 1055.0 mg·dm⁻³. The average content of total suspended solids (TSS) in wastewater in the filter bed was significantly higher than the value in raw wastewater indicated by Barka et al. [42]. These elevated TSS levels suggest two primary possibilities: (1) inadequate maintenance of the septic tank (component 2 in Figure 1), leading to carryover of settleable solids, or (2) washout of accumulated material from the soil-plant bed itself. The temporal variability likely reflects episodic events such as irregular septic tank emptying, variations in household water use patterns, or bed disturbance during maintenance. While we cannot rule out measurement error for the single extreme value (3031 mg·dm⁻³), the analytical procedure (EN 872:1996 gravimetric method) is robust, and samples were processed following strict protocols. Importantly, despite these high TSS values, the nematode community remained stable (as indicated by CCA analysis showing total nematode abundance in the centre of the ordination space), demonstrating the resilience of the bed biocenosis.

The concentration of phosphate phosphorus (P-PO₄) in the wastewater flowing through the bed ranged from 0.5 to 23.1 mgP-PO₄·dm⁻³. The average value was 9.9 mgP-PO₄·dm⁻³. The concentration of phosphorus pentoxide (P₂O₅) ranged from 1 to 53.1 mgP₂O₅·dm⁻³. The average value was 22.8 mgP₂O₅·dm⁻³. The concentration of total phosphorus (TP) in wastewater flowing through the bed ranged from 12.5 to 25.3 mgP·dm⁻³. The average value was 19.4 mgP·dm⁻³. The average content of total phosphorus (TP) in wastewater in the soil-plant bed was significantly higher than the value in raw wastewater, as indicated by Barka et al. [42]. The concentration of total nitrogen (TN) in wastewater flowing through the bed ranged from 111.7 to 145.8 mgN·dm⁻³. The average value was 131.9 mgN·dm⁻³, and the standard deviation (SD) was quite low, amounting to 18.

The concentration of ammonium nitrogen (N-NH₄) in wastewater flowing through the bed was in a wide range from 6.6 to 132.8 mgN-NH₄·dm⁻³. The average value was 68.8 mgN-NH₄·dm⁻³ and it was higher than the value in raw wastewater indicated by Barka et al. [42]. The concentration of nitrate nitrogen (N-NO₃) in wastewater flowing through the bed ranged from 7.6 to 26.7 mgN-NO₃·dm⁻³. The average value was 15.5 mgN-NO₃·dm⁻³. The concentration of nitrite nitrogen (N-NO₂) in wastewater flowing through the soil-plant bed ranged from 0.5 to 2.1 mgN-NO₂·dm⁻³. The average value was 1.4, and the standard deviation (SD) was very low, amounting to 1.0. The concentration of chlorides (Cl) in wastewater flowing through the soil-plant bed ranged from 129.9 to 240.9 mgCl·dm⁻³. The average value was 199.4 mgCl·dm⁻³. The concentration of iron (Fe) in wastewater flowing through the bed was 0.1 mgFe·dm⁻³ in each measurement period.

The abundance of nematodes inhabiting the filter bed in the Vertical Flow Constructed Wetland (VFCW) in Jabłonowo-Wypychy 10 ranged from 567,000.0 to 1,466,000.0 N·m⁻², with an average abundance of 1,064,666.7 N·m⁻². It should be noted that the standard deviation (SD) was very high, over 450,000. This means that the number of nematodes had a vast range. The largest trophic group were bacterivorous nematodes and fungivorous nematodes. The average abundance of bacterivorous nematodes was 738,730.0 N·m⁻². The average abundance of fungivorous nematodes was 240,033.3 N·m⁻². No predatory nematodes were detected in the soil and plant bed.

3.2. Physicochemical Parameters of Wastewater Flowing Through the Soil-Plant Bed and the Abundance and Trophic Groups of Nematodes Inhabiting This Bed in Rzące 25

The results of physicochemical tests of wastewater flowing through the soil-plant bed in the Rzące 25 Vertical Flow Constructed Wetland, as well as the abundance and trophic groups of nematodes in this bed, are presented in

Table 4. Concentration values of physicochemical parameters in sewage flowing through the soil-plant bed in Rzące 25.

St.							Av.	SD	CV [%]
pH	[-]	7.7	7.1	7.3	7.3	7.4	7.4	0	0
Temp	[°C]	4.8	18.0	19.4	5.3	19.0	13.3	8	60
DO	[mgO2·dm−3]	1.9	2.1	1.4	1.5	1.7	1.7	0	0
Alk	[mmol·dm−3]	10.2	10.1	173.0	2.4	10.3	41.2	74	180
BOD	[mgO2·dm−3]	102.0	78.5	97.5	68.0	30.0	75.2	29	38
COD	[mg·dm−3]	323.4	338.7	142.5	190.9	61.7	211.4	119	56
TSS	[mg·dm−3]	65.0	22.0	52.5	55.0	511.5	141.2	208	147
P-PO4	[mgP-PO4·dm−3]	5.3	4.3	9.3	7.2	0.2	5.3	3	57
P2O5	[mgP2O5·dm−3]	12.3	10.0	21.5	16.7	0.7	12.2	8	65
TP	[mgP·dm−3]	6.1	9.7	9.7	7.5	7.9	8.2	2	24
TN	[mgN·dm−3]	58.7	65.0	73.9	81.1	66.2	69.0	9	13
N-NH4	[mgN-NH4·dm−3]	0.8	6.0	0.9	11.8	2.1	4.3	5	116
N-NO3	[mgN-NO3·dm−3]	0.1	10.2	0.1	4.8	2.3	3.5	4	114
N-NO2	[mgN-NO2·dm−3]	140.2	142.4	175.9	64.1	45.1	113.5	56	49
Cl	[mgCl·dm−3]	0.2	0.3	0.2	0.2	0.2	0.2	0	0
Fe	[mgFe·dm−3]	0.8	6.0	0.9	11.8	2.1	4.3	5	116

Notes: St.—statistical analysis, Av.—average, SD—standard deviation, CV—coefficient of variation.

and

Table 5. Abundance and trophic groups of nematodes in soil-plant bed in Rzące 25.

St.	Nematode Abundance	Bacterivor.	Pred.	Fungivor.	Herbivor.	Omnivor.
	[N·m−2]
	375,000.0	236,250.0	11,250.0	131,250.0	0.0	0.0
	783,000.0	477,630.0	0.0	289,710.0	15,660.0	0.0
	539,000.0	334,180.0	16,170.0	167,090.0	21,560.0	0.0
	240,000.0	168,000.0	0.0	64,800.0	0.0	7200.0
	1,092,000.0	884,520.0	0.0	141,960.0	65,520.0	0.0
Av.	605,800.0	420,116.0	5484.0	158,962.0	20,548.0	1440.0
SD	338,870	284,488	7708	82,287	26,888	3220

Notes: Bacterivor.—bacterivorous, Pred.—predatory, Fungivor.—fungivorous, Herbivor.—herbivorous, Omnivor.—omnivorous.

. The pH of the wastewater flowing through the soil-plant bed in the hydrophytic wastewater treatment plant with vertical flow in Rzące 25 was neutral. Only one result was 7.7 and indicated an alkaline reaction of the wastewater. The average temperature (Temp) of the wastewater flowing through the bed was 13.3 °C and was lower than the temperature of the wastewater flowing through the soil and vegetation bed in the Jabłonowo-Wypychy 10 treatment plant.

The average concentration of dissolved oxygen (DO) in wastewater was 1.7 mgO₂·dm⁻³ and was similar to the concentration of dissolved oxygen (DO) in wastewater flowing through the soil and plant bed at the Jabłonowo-Wypychy 10 treatment plant. The average alkalinity (Alk) of the wastewater was 41.2 mmol·dm⁻³ and was much higher than the alkalinity (Alk) of the wastewater flowing through the soil and vegetation bed at the Jabłonowo-Wypychy 10 treatment plant. The mean value of biochemical oxygen demand (BOD) in wastewater was 75.2 mgO₂·dm⁻³ and was lower than the value of biochemical oxygen demand (BOD) in wastewater flowing through the soil and plant bed at the Jabłonowo-Wypychy 10 treatment plant.

The average value of chemical oxygen demand (COD) in wastewater flowing through the filter bed in the Rzące 25 treatment plant was also lower than the value of chemical oxygen demand (COD) in the wastewater flowing through the bed in the Jabłonowo-Wypychy 10 treatment plant. The average concentration of total suspension (TSS) in wastewater was 141.2 mgO₂·dm⁻³ and was significantly lower than the concentration of total suspension (TSS) in wastewater flowing through the soil and vegetation bed at the Jabłonowo-Wypychy 10 treatment plant.

The average concentration of phosphate phosphorus (P-PO₄) in wastewater was 5.3 mgP-PO₄·dm⁻³ and was lower than the value of this parameter in wastewater flowing through the filter bed at the Jabłonowo-Wypychy 10 treatment plant. The average concentration of phosphorus pentoxide (P₂O₅) in wastewater was 12.2 mgP₂O₅·dm^−3, and also the value of this parameter was lower than the value of phosphorus pentoxide (P₂O₅) in the wastewater flowing through the bed at the Jabłonowo-Wypychy 10 treatment plant. The average concentration of total phosphorus (TP) was 8.2 mgP·dm⁻³ and was lower than the concentration of this parameter in the wastewater flowing through the bed at the Jabłonowo-Wypychy 10 treatment plant.

The situation was similar with the concentration of total nitrogen (TN), ammonium nitrogen (N-NH₄) and nitrate nitrogen (N-NO₃) in wastewater. The average concentration of total nitrogen (TN) was 69.0 mgN·dm⁻³ and was lower than the value of this parameter in the wastewater flowing through the soil and vegetation bed in the Jabłonowo-Wypychy 10 treatment plant. The average concentration of ammonium nitrogen (N-HN₄) was 42.7 mgN-NH₄·dm⁻³ and was lower than the value of this parameter in the wastewater flowing through the soil and plant bed at the Jabłonowo-Wypychy 10 treatment plant. The average concentration of nitrate nitrogen (N-NO₃) was 4.3 mgN-NO₃·dm⁻³ and was also lower than the value of this parameter in the wastewater flowing through the soil and plant bed at the Jabłonowo-Wypychy 10 treatment plant. In the case of nitrite nitrogen (N-NO₂), the concentration of this parameter was higher in the wastewater flowing through the soil and plant bed in the Rzące 25 treatment plant.

The average concentration of chlorides (Cl) was 113.5 mgCl·dm⁻³ and was lower than the concentration of this parameter in the wastewater flowing through the soil and plant bed in the Jabłonowo-Wypychy 10 treatment plant. The abundance of nematodes inhabiting the soil-plant bed in Vertical Flow Constructed Wetland (VFCW) in Rzące 25 ranged from 240,000.0 N·m⁻² to 1,092,000.0 N·m^−2, and the average abundance was 605,800 N·m⁻². Standard deviation (SD) was very high, over 330,000. Average nematode abundance was higher in the soil-plant bed of the Jabłonowo-Wypychy 10 Vertical Flow Constructed Wetland (VFCW) than in the soil-plant bed of the Rzące 25 Vertical Flow Constructed Wetland (VFCW). The largest trophic group were bacterivorous nematodes and fungivorous nematodes. The average abundance of bacterivorous nematodes was 738,730.0 N·m⁻². The average abundance of fungivorous nematodes was 240,033.3 N·m⁻². Also, studies conducted by Atira and Kakouli-Duarte [13] showed that the most numerous trophic group was bacterivorous nematodes, because they were the most resistant to changing conditions in soil fertilised with different types of fertilisers.

The smallest trophic groups were herbivorous, predatory, and omnivorous nematodes. These results are consistent with those of Ghanem et al. [43], who reported a decrease in the abundance of herbivorous and predatory nematodes following soil-sewage contact. These two trophic groups of nematodes were found to be the most sensitive to wastewater contaminants.

Although detailed operational history and soil physicochemical properties were not systematically quantified in this study (a limitation we now acknowledge), several factors likely contributed to observed patterns: (1) Vegetation composition and root architecture in the soil-plant beds create distinct rhizosphere environments affecting microbial communities, which in turn influence bacterivorous and fungivorous nematode populations. (2) Bed age and organic matter accumulation history differ between sites, potentially affecting food web complexity and soil structure. (3) Maintenance practices (frequency of bed surface disturbance, vegetation management) may influence nematode community composition. (4) The near absence of predatory nematodes at both sites, consistent with findings by Ghanem et al. [43] cited in our manuscript, suggests that wastewater pollutants exert selective pressure against these sensitive trophic groups, though their occasional detection at Rzące 25 indicates some tolerance variability. We have added a dedicated paragraph discussing these ecological and operational factors in the Results and Discussion section, and note that future research systematically documenting these variables would further elucidate mechanisms.

3.3. Canonical Correspondence Analysis (CCA)

The results of canonical correspondence analysis (CCA) for the soil-plant deposit in the Jabłonowo-Wypychy 10 Vertical Flow Constructed Wetland (VFCW) are presented in Figure 2.

The CCA analysis for the soil-plant bed in the Jabłonowo-Wypychy 10 Vertical Flow Constructed Wetland (VFCW) showed that the total abundance of nematodes (All_Nematodes) was located in the centre of the CCA graph, which suggests that the strength of the influence of all physicochemical parameters of wastewater on the total abundance of nematodes (All_Nematodes) was the same. None of the physicochemical parameters of wastewater showed a more substantial effect on the total abundance of nematodes (All_Nematodes).

The abundance of bacterivorous nematodes was strongly correlated with dissolved oxygen (DO), biochemical oxygen demand (BOD), pH, temperature (Temp), and total suspended solids (TSS). For the fungivorous nematodes in the soil-plant bed in Jabłonowo-Wypychy 10 Vertical Flow Constructed Wetland (VFCW), the concentration of chemical oxygen demand (COD) was a significant differentiating factor.

The abundance of herbivorous nematodes was correlated with concentration of nitrate nitrogen (N-NO₃) and concentration of nitrite nitrogen (N-NO₂). For the omnivorous nematodes, the concentration of nitrate nitrogen (N-NO₃) and nitrite nitrogen (N-NO₂) was also a significant differentiating factor. The results of canonical correlation analysis (CCA) for the soil-plant deposit in the Rzące 25 Vertical Flow Constructed Wetland (VFCW) are presented in Figure 3.

The CCA analysis for the soil-plant bed in the Rzące 25 Vertical Flow Constructed Wetland (VFCW) also showed that the total abundance of nematodes (All_Nematodes) was located in the centre of the CCA graph, which suggests that the strength of the influence of all physicochemical parameters of wastewater on the total abundance of nematodes (All_Nematodes) was homogeneous. Despite the observed variability in the response of individual trophic groups to environmental factors, the total nematode abundance (All_Nematodes) remained relatively stable—as evidenced by its central position in the CCA analysis graphs. This result suggests that the total number of soil nematodes is insensitive to minor changes in wastewater composition, indicating a high resistance of the entire biocenosis to variability in the pollutant load.

The stability of total nematode abundance across variable wastewater input conditions (as evidenced by CCA showing “AllNematodes” positioned centrally in ordination space at both sites) demonstrates ecological resilience—the ability of the soil biocenosis to maintain functional capacity despite environmental fluctuations. This resilience contributes to VFCW performance stability through several mechanisms: (1) Nutrient cycling continuity—bacterivorous nematodes enhance nitrogen mineralisation by grazing bacteria, making nutrients available for plant uptake; their stable abundance ensures this process continues despite wastewater quality variations. (2) Microbial community regulation—nematode grazing prevents bacterial over growth and maintains diverse microbial communities capable of degrading various organic compounds. (3) Soil structure maintenance—nematode movement through soil pores helps maintain hydraulic conductivity. (4) Functional redundancy—while specific trophic group abundances vary with conditions, the total community maintains sufficient functional diversity to support treatment processes. This ecological resilience is particularly valuable for small-scale, low-maintenance systems serving individual households where wastewater inputs are inherently variable.

The abundance of bacterivorous nematodes was mainly associated with total suspended solids (TSS), ammonium nitrogen (N-NH₄), temperature (Temp), total nitrogen (TN), and total phosphorus (TP).

From the above, it follows that the same physicochemical parameters of wastewater, on which the number of bacterivorous nematodes in both deposits depended, were total suspension (TSS) and wastewater temperature (Temp). Therefore, it is crucial to regularly empty the settling tank, which affects the concentration of total suspended solids (TSS) in the wastewater.

For the fungivorous nematodes in the soil-plant bed in Rzące 25 Vertical Flow Constructed Wetland (VFCW), the concentration of chemical oxygen demand (COD), biochemical oxygen demand (BOD), phosphorus pentoxide (P₂O₅), phosphate phosphorus (P-PO₄), nitrite nitrogen (N-NO₂), nitrate nitrogen (N-NO₃), iron (Fe), chlorides (Cl), dissolved oxygen (DO), alkalinity (Alk), and pH were significant differentiating factors.

A standard parameter for both deposits was the chemical oxygen demand (COD). Therefore, the number of fungivorous nematodes depends on the content of organic substances and some inorganic substances in the bed.

The abundance of herbivorous nematodes was correlated with temperature (Temp), concentration of total suspended solids (TSS), and concentration of total phosphorus (TP).

At Jabłonowo-Wypychy 10, bacterivorous nematode abundance was strongly correlated with dissolved oxygen, BOD, pH, temperature, and TSS, while at Rzące 25, they responded primarily to TSS, ammonium nitrogen, temperature, total nitrogen, and total phosphorus. These site-specific patterns likely reflect: (1) Microbial community composition—bacterivorous nematodes feed on bacteria, so differences in bacterial community structure (driven by local soil conditions and organic matter quality) would affect nematode food availability and trophic preferences. (2) Plant species and root architecture—different marsh vegetation establishes distinct rhizosphere micro environments with varying oxygen availability, root exudates, and bacterial populations. (3) Bed maturity and organic matter history—older beds may have accumulated organic matter differently, affecting decomposition dynamics and bacterial communities. (4) Hydraulic loading patterns—household wastewater discharge patterns and infiltration rates influence oxygen distribution and nutrient availability. These factors interact to create site-specific “rhizosphere effects” that mediate how wastewater parameters translate into bacterivorous nematode responses.

In the soil-plant bed of the Rzące 25 Vertical Flow Constructed Wetland (VFCW), no effect of any of the physicochemical variables tested on omnivorous nematodes’ abundance was observed. The abundance of predatory nematodes was correlated with the concentration of chlorides (Cl), iron (Fe), biochemical oxygen demand (BOD), chemical oxygen demand (COD), dissolved oxygen (DO), pH, and alkalinity (Alk). The obtained research results indicated environmental factors (including iron-heavy metal) influencing the number of predatory nematodes, which are characterised by very high sensitivity to pollutants contained in sewage [41].

3.4. Mann–Whitney–Wilcoxon Test

Table 6. Results of Mann–Whitney–Wilcoxon analysis for effluent flowing into soil-plant beds in Vertical Flow Constructed Wetland in Jabłonowo-Wypychy 10 and Vertical Flow Constructed Wetland in Rzące 25 [own elaboration].

Ordinal Number	Physicochemical Parameter	Parameter “W”	Parameter “p-Value”	Statistical Significance
1.	Alkalinity (Alk)	23	0.283	absence
2.	Biochemical oxygen demand (BOD)	10	0.3677	absence
3.	Chlorides (Cl)	24	0.2141	absence
4.	Chemical oxygen demand (COD)	16	1	absence
5.	Iron (Fe)	4	0.0448	absence
6.	Ammonium nitrogen (N-NH4)	19	0.6706	absence
7.	Nitrite nitrogen (N-NO2)	18	0.7763	absence
8.	Nitrate nitrogen (N-NO3)	27.5	0.060	absence
9.	Total nitrogen (TN)	22	0.3677	absence
10.	Dissolved oxygen (DO)	7	0.1453	absence
11.	Phosphorus pentoxide (P2O5)	18	0.8081	absence
12.	pH reaction	16.5	1	absence
13.	Phosphate phosphorus (P-PO4)	21.5	0.3949	absence
14.	Total phosphorus (TP)	24	0.2141	absence
15.	Total suspended solids (TSS)	10	0.3677	absence
16.	Temperature (Temp)	23	0.2828	absence

shows the results of the statistical analysis of the Mann–Whitney–Wilcoxon test, which aimed to test for statistically significant differences between successive parameters of the effluent flowing into the soil-plant beds at the analysed wastewater treatment plants.

The statistical analysis results showed that for most physicochemical parameters studied, no statistically significant differences were observed between the effluent samples flowing into bed one and bed two (all p > 0.05). Although a statistically significant difference (p < 0.05) was found for iron (Fe), complementary analyses did not confirm this result, as it showed no sustained effect on the structure of the trophic assemblages, and was therefore not considered ecologically significant.

The absence of statistically significant differences suggests that the composition of the effluent flowing into the two VFCW deposits was homogeneous in terms of the physicochemical characteristics analysed, which increases the comparability of the results of the biotic analyses and the reliability of the observed trophic relationships in nematode structure.

4. Conclusions

After 15 years of operation at the sewage treatment plant, soil-plant deposits were heavily colonised by soil nematodes. Bacterivorous nematodes constituted the most numerous trophic group.

Although the physicochemical parameters of the sewage flowing into both VFCW beds were significantly similar and their differences were not statistically significant, the CCA analysis revealed significant differences in sewage parameters affecting the abundance of various trophic groups of soil nematodes in each bed.

The reason for the response of different trophic groups of nematodes to different sets of variables may be the way the wastewater treatment plant is operated, changes in the construction of the treatment plant that have occurred spontaneously over the last 15 years, or different vegetation growing in the deposits.

The total abundance of soil nematodes was not sensitive to small changes in sewage composition, which indicates a high resistance of the entire biocenosis to changes in pollutant load.

Based on the data obtained, it can be concluded that possible disruptions in the operation of the treatment plant—e.g., periodic deterioration in the quality of wastewater related to the lack of emptying of the settling tank, excessive load or reduction of flow—do not lead to a decrease in the overall number of nematodes. However, they may result in a change in the proportion of trophic groups, which may indirectly affect the functioning of the deposit ecosystem. The ability of the deposit to maintain biological activity despite changing environmental conditions demonstrates its high ecological resilience and ability to self-regulate, which should be considered one of the main advantages of nature-based treatment systems (NBS). In the light of the results obtained, VFCW deposits show the potential for long-term and stable operation even in the event of disturbance episodes, which reinforces their value as elements of sustainable water and wastewater infrastructure.