Quantification, phase distribution, and persistence of antiretroviral drugs in wastewater treatment systems and their ecotoxicological impacts in aquatic environments
South Africa has the world’s largest antiretroviral therapy (ART) programme, treating more than 5.5 million people, which has resulted in high levels of antiretroviral drugs (ARVs) in wastewater and environmental waters. Despite this scale, the occurrence, fate, and ecological effects of ARVs in wastewater treatment plants (WWTPs) and receiving waters remain poorly understood. This study investigated three widely prescribed ARVs, efavirenz (EFZ), emtricitabine (EMT) and nevirapine (NVP), examining their behaviour in conventional activated sludge (CAS) WWTPs and their potential effects on aquatic organisms. Seasonal monitoring of two WWTPs over two years showed consistent detection of all three ARVs in influent and effluent. EMT occurred at the highest concentrations, followed by EFZ and NVP. Reduction efficiencies varied between plants and seasons, with EMT showing the most consistent removal, while EFZ and NVP frequently displayed low or negative values. Multivariate analysis revealed clear seasonal structuring and associations with routine water quality determinants, such as manganese, lead, magnesium, etc., indicating that operational and environmental factors influence ARV behaviour during treatment. Process-level investigations revealed compound-specific phase distributions driven by physicochemical properties: emtricitabine, characterised by high polarity and low sorption affinity, remained predominantly in the aqueous phase, efavirenz exhibited strong sorption to solids, and nevirapine, with intermediate polarity and ionisation behaviour, partitioned between aqueous and solid phases. Pharmaceuticals were not consistently reduced across treatment stages or over time, with negative stage-specific reduction efficiencies reflecting dynamic phase exchanges, including desorption and microbial deconjugation, rather than simple degradation. Ecotoxicological testing using a battery of bioassays revealed variable endocrine responses in yeast-based assays (YES, YAS, YAES and YAAS), low bacterial and algal sensitivity (Aliivibrio fischeri bioluminescence inhibition and Raphidocelis subcapitata algal growth inhibition assays), and stronger effects at higher trophic levels (Daphnia magna immobilisation and Danio rerio fish embryo toxicity tests). Behavioural endpoints, such as changes in zebrafish larval activity, were the most sensitive indicators of ARV impact, identifying effects at concentrations below the detection threshold of standard ecotoxicity assays. These effects were observed at concentrations comparable to those measured in South African surface waters, while conventional mortality and morphological endpoints did not indicate any effects at these levels. The combined findings demonstrate that ARVs persist throughout CAS treatment systems, exhibit compound-specific behaviour, and can exert sub-lethal biological effects at environmentally relevant concentrations. This highlights the limitations of existing treatment infrastructure and the reliance on routine chemical monitoring alone. This research provides evidence-based recommendations for integrating effect-based tools such as behavioural bioassays with chemical monitoring and for applying compound-specific treatment strategies. Although this work was conducted within a South African context, its insights extend to other high-burden regions facing similar challenges. These findings support the development of more effective and proactive approaches to managing pharmaceutical contamination in aquatic environments.
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