Biofilm resilience in a constructed wetland: Response to hexachlorocyclohexane

Organochlorine contamination by hexachlorocyclohexane (HCH) and chlorobenzenes (ClB), co-occurring with iron and manganese, poses persistent challenges for constructed wetland treatment systems. Microbial biofilms are central to pollutant attenuation, yet the responses of benthic diatoms and bacterial communities to such complex chemical gradients remain poorly understood. Biofilm communities were sampled on standardised fritted glass disc carriers ( n = 15 per site, 25 days incubation) and natural substrates ( n = 6 per site) at five sites spanning the biosorption/biodegradation (module C) and aerobic wetland (module D) compartments of a full-scale modular treatment system (Wetland+, Hájek, Czech Republic). Metabolic function (net primary production, community respiration), phototrophic biomass (chlorophyll-a), diatom assemblages (microscopy, Shannon diversity) and bacterial communities (16S rRNA amplicon sequencing) were characterised alongside hydrochemical and contaminant profiling. Biofilm metabolism was predominantly heterotrophic (NPP:CR < 1) at all sites, with strongest suppression at upstream sites where HCH (12.25–13.35 μg/L), ClB and Fe concentrations were highest. Chlorophyll-a and net primary production increased downstream as pollutant loads declined. Benthic diatoms dominated the phytobenthos throughout; 48 taxa across 21 genera were identified, with species richness increasing from 4 to 5 taxa at upstream sites to 23–24 at downstream sites. Key tolerant taxa included Achnanthidium minutissimum , Rhopalodia parallela and Nitzschia palea . Bacterial communities clustered distinctly by module: Parasediminibacterium and Rhodoferax dominated module C, while Rhodobacteraceae and diatom diversity increased in module D. Fritted glass carriers supported diatom assemblages comparable to, or richer than, natural substrates at most sites. These findings demonstrate a clear pollution-driven gradient in biofilm structure and function across the Wetland+ system. The integration of metabolic measurements, diatom assemblages and bacterial community profiling provides a robust framework for evaluating organochlorine remediation performance, supporting the use of standardised biofilm carriers for ecological monitoring in constructed wetlands.