## 2 Materials and Methods
### Study Area
Figure 2: Map of the Zaalklapspruit study area and sampling sites within the Olifants River drainage system in South Africa (adapted from the South African National Biodiversity Institute (SANBI) [35]). This study employed an influent-effluent comparison method for examining wetland water remediation, which typically regards the ecosystem as a \"black box\". This approach provides a constrained view of the biogeochemical processes occurring within the wetland [40]. Previous research has shown that slowing the water flow in natural wetlands enhances the water residence time, thereby improving sediment infiltration and the interaction between AMD contaminants and various wetland components, such as bottom sediment [3; 8; 41]. This study focused on exploring the ability of the wetland's bottom sediment and rhizosphere to accumulate metals and sulfate. Sampling took place in the dry season of May 2022 to reduce the influence of seasonal rainfall, which could potentially modify the surface water flows, groundwater levels, and AMD concentrations [28; 42]. A systematic sampling strategy was employed, adhering to the guidelines set by the [43], involving the careful selection of three study sites within the wetland. These sites were strategically chosen based on their distance from the wetland inflow and were designated as follows: inflow = Site 1 (S1), middle = Site 2 (S2), and far middle = Site 3 (S3). Site S1, closest to the inflow, received surface water most directly impacted by AMD effluent; Site S2 experienced intermediate exposure to the wetland's remedial processes; and Site S3, the farthest away, was influenced by these processes for the longest duration. The sampling regimen included the collection of both abiotic (surface water, wetland sediment, and surface soil precipitate) and biotic (macrophyte root systems and periphyton species) components. Samples were collected in triplicate (n = 3) using a simple randomized sampling method as recommended by the [43] to ensure a comprehensive and unbiased evaluation of the sites. All the collected samples were promptly sealed, stored in cool, dry containers, and transported to accredited laboratories within 24 h for analysis. These samples were analyzed for a variety of characteristics using the specific methods detailed in Table 1.  
Figure 3: Concrete structures and weirs to reduce the surface water flow in the ecologically engineered wetland, Zaalkapspruit, Mpumalanga, South Africa (2022). Table 1 summarizes the substrates sampled and the corresponding analytical methods used for each assessment. Surface water was analyzed for physical and chemical characteristics using electrometric, conductimertic, and ICP spectrometry methods. The bottom sediment underwent elemental composition analysis via X-ray fluorescence (XRF), texture analysis using the pipette method, and microbiological diversity assessment through 16s RNA sequencing. Macrophage roots and periphyton were examined for metal adsorption and absorption using ICP-OES, while unflooded soil precipitate was analyzed for mineralogy also using XRF. These methods collectively provided a detailed understanding of the wetland's physical, chemical, and biological attributes, supporting a thorough evaluation of its condition and the effectiveness of AMD remediation efforts.