Unsafe drinking water is a major cause of human disease, particularly in low-income countries lacking centralized water treatment systems. Chlorine, commonly used as sodium hypochlorite (NaOCl), effectively disinfects water but produces undesirable disinfection by-products (DBPs). Point-of-use (POU) systems offer a decentralized alternative, eliminating the need for residual chlorine in distribution networks. The World Health Organization recommends free chlorine concentrations of 0.2–0.5 mg L⁻¹ at the point of use. Conventional chlorine-based disinfectants in POU systems necessitate the handling of hazardous chemicals and can lead to DBP formation, impacting taste and odor. Ultraviolet and ozone are established disinfection technologies, but electrochemically activated solutions (ECAS) offer the advantage of external application in various settings, including food production and healthcare. Limited studies have directly compared ECAS with common chlorine agents for decentralized disinfection, with insufficient information on ECAS pH and biofilm effectiveness. Electrochemical disinfection technologies, already established in food and healthcare, generate ECAS by passing a saline solution through an electrochemical cell. Anodic solutions are highly oxidative, resulting in acidic pH (2–5) and antimicrobial species dominated by HOCl. The antimicrobial action involves disrupting cell membranes, affecting microbial functionality. Most microorganisms exist in biofilms, which provide protection against disinfectants. Biofilms in water systems cause biofouling and act as reservoirs for pathogens, increasing antimicrobial resistance. This study aimed to compare the antimicrobial and biofilm-inhibiting properties of OCl⁻, HOCl, and ECAS for POU drinking water applications, using *E. coli* in bactericidal assays and *P. aeruginosa* in biofilm assays.

The literature review extensively covered the prevalence of waterborne diseases, particularly in low-income countries, highlighting the need for effective and safe water disinfection methods. Existing centralized systems using chlorine often lead to the formation of harmful byproducts. The advantages and limitations of alternative disinfection technologies like UV and ozone were discussed. The literature also covered the use of electrochemically activated solutions (ECAS) in various settings, highlighting the potential of this technology for water disinfection. Studies comparing ECAS to other chlorine-based disinfectants were reviewed, pointing out the knowledge gap regarding ECAS's effect on biofilms and the specific chemical species involved in its antimicrobial action. Finally, the role of biofilms in drinking water systems and their resistance to disinfectants was explored, emphasizing the need to investigate the efficacy of disinfectants against biofilms.

Three chlorine-based disinfectants were tested: NaOCl (dominant species OCl⁻), HOCI (neutral and slightly acidic), and electrochemically generated acidic HOCl (ECAS). Stock solutions were prepared and standardized to specific free chlorine (FC) concentrations. Antimicrobial activity against planktonic *E. coli* was assessed using BS EN 1040 (no organic load) and BS EN 1276 (with organic load – bovine serum albumin [BSA] at 0.3 g L⁻¹ (clean) and 3.0 g L⁻¹ (dirty)) assays. A 5-log reduction was the threshold for bactericidal activity. Anti-biofilm activity against established *P. aeruginosa* biofilms was determined using a CDC biofilm reactor. Biofilms were grown on polycarbonate coupons and exposed to various FC concentrations for 5 minutes. Biofilm density was measured by colony counting after sonication. CT values (concentration × time) were calculated for 5-log reduction of *E. coli* and 2-log reduction of *P. aeruginosa* biofilms. Statistical analysis (two-way ANOVA with Tukey's post-test) was used to compare the antimicrobial activity of the disinfectants. The pH and ORP of the solutions were measured.

All disinfectants showed significant antimicrobial activity against planktonic *E. coli*. In the absence of organic load, all disinfectants achieved a 5-log reduction at ≥50 mg L⁻¹ FC. At 25 mg L⁻¹ FC, HOCI showed the highest activity. The presence of a low organic load (0.3 g L⁻¹ BSA) affected the activity of all disinfectants, but ECAS maintained ≥5-log reduction at 25 mg L⁻¹. A high organic load (3.0 g L⁻¹ BSA) significantly reduced the activity of OCl⁻ and ECAS. HOCI exhibited the highest activity at all FC concentrations tested under this high organic load condition. For *P. aeruginosa* biofilms, ECAS showed the greatest reduction in biofilm density at all FC concentrations ≥50 mg L⁻¹. OCl⁻ and HOCl showed significantly lower reductions. At FC concentrations below 50mg L⁻¹, no significant difference was observed between the disinfectants in terms of anti-biofilm activity. CT values for a 5-log reduction of planktonic *E. coli* and a 2-log reduction of *P. aeruginosa* biofilms were calculated. HOCI had the lowest CT value for planktonic *E. coli* in the absence of organic load (16.51 mg min L⁻¹), while ECAS showed the lowest CT value with a clean BSA load (20.94 mg min L⁻¹). ECAS showed the lowest CT value for *P. aeruginosa* biofilm reduction (87.21 mg min L⁻¹).

The findings demonstrate that HOCI is the most effective against planktonic *E. coli*, aligning with its ability to penetrate cell membranes. The reduced efficacy of OCl⁻ is attributed to its limited membrane penetration. The impact of organic matter highlights the differing mechanisms of action. ECAS, with its high ORP and multiple active species, shows resilience against low organic loads. The higher organic load reduced the efficacy of all disinfectants, demonstrating the importance of pre-treatment to remove organic matter. The superior anti-biofilm activity of ECAS is attributed to its high ORP, disrupting the EPS matrix and enabling penetration of bacterial cells by HOCl. The differences in efficacy between planktonic and biofilm disinfection highlight the importance of considering both aspects when selecting a disinfectant for POU systems. Previous studies supported the minimal trihalomethane formation with HOCI and ECAS, unlike OCl⁻, making them safer alternatives in systems lacking pre-treatment. The ability to generate ECAS in situ reduces the need for hazardous chemical storage and transportation.

This study comprehensively evaluated three chlorine-based disinfectants for POU water treatment. HOCI proved most effective against planktonic bacteria, while ECAS demonstrated superior anti-biofilm activity. ECAS's in-situ generation offers safety and logistical advantages, minimizing risks associated with chemical handling. However, future studies should investigate multi-species biofilms and the impact of different materials to fully understand the efficacy and suitability of these disinfectants in real-world POU systems. Monitoring residual free chlorine concentrations post-disinfection is essential to optimize treatment parameters and ensure compliance with WHO guidelines.

The study used single-species cultures for both planktonic and biofilm assays, which may not fully represent the complexity of real-world water sources. The organic loading used, while informative, may not encompass the full range of organic matter present in various water sources. Residual free chlorine concentrations were not monitored post-treatment, potentially limiting the interpretation of results in terms of actual disinfectant residual levels. Further studies are needed using more complex model systems that incorporate diverse microbial communities and variations in water matrix composition.