*4.1. Lab-Scale*

Scientists in Portugal conducted laboratory-scaled microcosm studies to evaluate the removal performance of constructed wetlands for veterinary antibiotics for many years [39,42,73]. In their CW microcosms, multiple layers were set up (from bottom to top) as gravel, lava rock, root bed substrate (which was a mixture of soil and sand to help the vegetation's establishment) and *Phragmites australis* were planted. They used wastewater from swine farms/saline aquaculture facilities as their influent water with antibiotic concentrations spiked-up to 100 μg/L. The results showed that the removal efficiency for vet antibiotics-enrofloxacin (ENR), tetracycline (TET) [39], oxytetracycline (OXY) [73] and ceftiofur (CEF) [42] were over 90% after 9 to 20 weeks treatment period. The major mechanisms for the removal processes were adsorption to the substrate and plant's root (physical process), microbial metabolization and degradation (biological and chemical processes) and plant uptake (biological process) [39,42,73]. Studies conducted using the wide range of pollutants and various influent water types (fresh water and saline water) proved that CW microcosm design was adaptable to various wastewater treatments with satisfying removal efficiencies. Their study in 2020 using the same system even achieved toxic metal removal while maintaining the nutrient levels for agriculture reuse [70]. Another study in 2018 using the same system observed removal of organic micropollutants, such as atrazine, clarithromycin, fluoxetine, and norfluoxetine, from the freshwater aquaculture effluents [74]. Evidence from other studies suggested that such removal was accomplished through microbial degradation [75,76]. Another study conducted in Canada also found that subsurface horizontal flow constructed wetlands could remove 42%, 49% and 49%, respectively, of poultry pharmaceuticals monensin, salinomycin and narasin through sorption onto the soil surface and microbial degradation [77]. This indicates that with successful CW design, we can treat wastewater containing various contaminants in an efficient and economical manner. Such small-scale laboratory studies may not be sufficient for direct field application of constructed wetlands, but they serve as a good role at the proof-of-concept stage for future larger scaled studies.

Besides antibiotics, pesticides, such as chlorpyrifos, have been studied intensively and shown to be highly removable through constructed wetlands [78–82]. Most of the studies showed that biodegradation and adsorption were the primary removal mechanisms of such chemicals from the CW system. In addition, studies have also looked into the removal performance for antibiotic resistance genes [83–85]. According to the study by Song et al. (2018), the accumulation of antibiotics in different layers within the constructed wetland resulted in an abundance of ARGs with a positive correlation relationship [84]. Later studies proved that some CW systems could reduce ARG concentrations as they remove the antibiotic contaminations. Chen et al. 2019 study showed that while antibiotics' major removal mechanism was microbial degradation, ARGs main removal mechanisms were substrate sorption and biological reactions [83]. Another study investigated the comparisons of substrate medium by Du et al. (2020) and observed better removal rates (>95%) for both antibiotics and ARGs when zeolite medium and plant (*Arundo donax*) were used [85].
