Photolysis of sodium chloride and sodium hypochlorite by ultraviolet light inactivates the trophozoites and cysts of Acanthamoeba castellanii in the water matrix

The present study aimed to investigate an effective, sustainable and accessible way to inactivate chlorine-resistant microorganisms, such as Acanthamoeba castellanii, through the photolysis of sodium chloride (NaCl) and sodium hypochlorite (NaOCl) in the water matrix. The trophozoites and cysts (2 × 10 per 8 mL) were exposed for 30, 60, 90, 120 and 150 min to the photolysis effect of NaOCl (1.0, 2.0, 4.0 and 8.0 mg/L) or NaCl (5.0, 10, 20 and 40 g/L) by ultraviolet light C (243 μW·cm), then the viability was analyzed. The inactivation of all trophozoites was achieved by exposure to the photolysis effect of 2.0 mg/L of NaOCl or 20 g/L of NaCl, in 150 or 120 min, respectively. Inactivation of all cysts was achieved by double exposure to the photolysis effect of 1.0 mg/L NaOCl or 5.0 g/L NaCl from 90 min of each exposure round. The exposure time was a strong determinant in the inactivation of A. castellanii trophozoites or cysts. The photolysis of NaOCl or NaCl is an effective method to eliminate A. castellanii in water. These findings expand the list of chlorine-resistant microorganisms that can be inactivated by NaOCl photolysis and show that NaCl photolysis is a new and promising method for treating swimming pool water and wastewater.

Amoebae exist as trophozoites, which are the active forms that feed and reproduce, or as cyst, which is the environmentally resistant form (Garajová et al. ).

).
Acanthamoeba is an opportunistic pathogen that has been increasingly reported in human pathologies, as in the case of AK, which is favored by the increase in contact lens wearers associated with some risky behavior (Nagington et  One of the potential ways to achieve inactivation of FLA in water is the use of advanced oxidation processes, such as photolysis (Remucal & Manley ). In the photolysis of disinfectants such as sodium hypochlorite (NaOCl), hypochlorous acid (HOCl) or hypochlorite (ClO À ), several reactive oxidants are produced, including OH · , O 3 and chlorine radical (Cl · ), that have biocidal effect (Buxton & Subhani ; Remucal & Manley ; Astuti et al. ).

The formation of transient forms of chlorine, including
Cl 2 , Cl À 3 , HClO À , ClO À and Cl À , has also been observed during the exposure of highly concentrated NaCl solutions to ionizing radiation, such as gamma radiation (Büppelmann In the present study, we evaluated the effect of sodium chloride (NaCl) and NaOCl photolysis by ultraviolet C radiation on the viability of trophozoites and cysts of A.
castellanii and demonstrated that the photolysis of NaCl or NaOCl in the water matrix is able to inactivate trophozoites and cysts.

Chemicals and materials
The growth media and reagents were purchased from commercial suppliers. Deionized water by reverse osmosis was used to prepare the growth media, which were autoclaved before use.

Disinfection procedures
The experiments were carried out in duplicates with three replications. Water with different doses of NaOCl and NaCl, previously contaminated with 2 × 10 7 trophozoites/8 mL or 10 7 cysts/8 mL of water, was exposed to ultraviolet C light (λ ¼ 254 nm). About 8 mL of water was placed in glass plates measuring 27 cm 2 of the surface area, and the body of water had a thickness of ∼1 cm. The plates were placed in a biological safety cabinet equipped with a UV-C lamp (11-W low-pressure Hg UV lamp with a wavelength of 254 nm, 4P-SE, Philips). The mean intensity of the measured UV-C was 243 μW·cm 2 using a UV radiometer (lux meter X1-1 UV-C radiometer, UV-3726 model) at 254 nm. The plates were placed at a distance of 60 cm from the lamp. The lamp was previously turned on for 15 min before the start of the experiment. The exposure time was 60, 90, 120 and 150 min for treatment with NaOCl and 30, 60, 90, 120 and 150 min for treatment with NaCl.
In the experiments with trophozoites, two control approaches were implemented: (i) the effect of NaOCl/ NaCl only and (ii) the effect of UV-C only. In experiments with cysts, a single control approach was performed, evaluating the effect of NaOCl/NaCl only. In our previous tests, we found that A. castellanii cysts were resistant to the isolated effect of UV-C, even after being exposed for 120 consecutive minutes (our unpublished data). However, a reduction of 6 logs was achieved when the water temperature was raised and kept at 55 C, then exposed to UV-C for 10 min, using procedures described in the literature (Heaselgrave & Kilvington ).
In the second stage of the experiment, the cysts were subjected to double exposure to the effect of NaOCl/NaCl photolysis in the water matrix by UV-C. After the first exposure, the cysts were kept in water and in the dark for 72 h, then the water was exposed to UV-C again. The water containing NaOCl received new doses of chlorine before the second exposure. In this stage, treatments such as 1.0, 2.0 and 4.0 mg/L for NaOCl and 5.0, 10 and 20 g/L for NaCl were implemented, with the same contact times as mentioned above.

Viability analysis of trophozoites and cysts
The entire volume of treated water was centrifuged (1,500 rpm for 5 min), discarding the supernatant. The pellet was washed twice with deionized water previously autoclaved (to remove residues of NaOCl or NaCl) and then resuspended in 100 μL. The viability analyses of trophozoites and cysts were performed using two different techniques.

Trophozoites
About 10 μL of 0.4% trypan blue dye was added to 100 μL of the suspension, then incubated for 5 min, and viable trophozoites were counted using a Fuchs Rosenthal counting chamber in an inverted optical microscope with phase contrast. Although the culture was also considered for viability analysis, it was not considered adequate, because during the exposure of trophozoites to the effect of NaOCl/NaCl photolysis, it was observed that the cells were fragmented. It was observed that the longer the exposure time to UV-C radiation of solutions with increasing concentrations of NaOCl/NaCl, less or no whole trophozoite cell was observed. For this reason, it was not possible to count the portion of inactivated trophozoites.

Cysts
Spots of 50 μL of cyst suspension were seeded in the center of each of the two quadrants of the fresh NNA plate on which a heat-inactivated Escherichia coli layer was previously spread. After spots drying, the plates were incubated for 9 days at 30 C and checked after 1, 2, 3, 6 and 9 days of incubation. Cysts exposed twice to the effect of NaOCl/NaCl photolysis in the water matrix by UV-C were checked for up to 12 days.
Five randomized microscopy fields (100×) were found on each NNA agar quadrant, in the area where the spot was deposited, to check for the absence or presence of trophozoites. If present, the number of trophozoites observed per microscopy field was counted and an arithmetic mean was calculated. Microscopy fields with higher density and homogeneous distribution of cysts or trophozoites were considered.
No mathematical model was used to relate the number of trophozoites observed per microscopic field, with the initial number of cysts. Our aim was to find out whether the cysts were viable after treatment and whether the number of trophozoites recovered from the cysts decreased with an increasing NaOCl/NaCl concentration and the time of exposure to UV-C. We also aim to determine the minimum necessary concentration of NaOCl/NaCl and the exposure time to UV-C required to inactivate 10 6 cysts/8 mL in the water matrix.

DATA ANALYSIS
The two-tailed paired t-test was used to determine the significance of the differences between the data using the BioEstat 5.0 software. A value of p < 0.05 was considered significant.
The GraphPad prism 8.02 program was used to plot graphs.

Single exposure
The results show that there was a slight reduction in the viability of cysts exposed to the isolated effect of NaOCl, but a drastic reduction was observed when they were subjected to the effect of NaOCl photolysis by UV-C in all treatments.
The average number of trophozoites recovered from the cysts differed significantly from the corresponding controls (NaOCl only) (p < 0.05) in most counts in all treatments.
However, inactivation of all cysts was not achieved ( Figure 5).

Double exposure
The results show that there was inactivation of all cysts in almost all treatments. The recovery of few trophozoites from the treated cysts occurred only in the 1.0 mg/L treatment with 60 min of exposure time ( Figure 6).

Single exposure
The data show a strong similarity with the data obtained in the tests with NaOCl. The means of trophozoites recovered from the treated cysts differed significantly from the means of the corresponding controls (p < 0.05) in most counts in all treatments. The inactivation of all cysts was not achieved in any of the treatments (Figure 7).

Double exposure
The data also closely resemble the data previously presented Our findings (Figures 5 and 6) show that the number of trophozoites recovered from treated cysts is more strongly influenced by the exposure time to the effect of NaOCl and NaCl photolysis by UV-C than by the concentration of FAC or NaCl in the water matrix (Tables 1 and 2).

DISCUSSION
To verify the effect of NaCl and NaOCl photolysis by UV on the viability of A. castellanii trophozoites and cysts in the water matrix, solutions containing with 0.5, 1.0, 2.0 and 4.0% of NaCl and NaOCl, previously contaminated by trophozoites or cysts, were exposed to UV-C for different contact times. Although the reduction in the number of viable trophozoites in deionized water was observed by exposure to the isolated effect of UV-C radiation, in general this was considerably greater in the NaCl and NaOCl solutions exposed to UV-C. In the NaOClþ UV-C treatment, at con-    immediately inactivate the trophozoites, but the cell damage was severe enough to induce cell death. This may also suggest that trophozoites exposed to the synergistic effect of oxidizing radicals and UV-C during NaOCl photolysis become more sensitive to the isolated effect of UV-C.
Our data show that the trophozoites were resistant to all concentrations of NaCl only at the different exposure times tested, although some slight reduction in viability was observed at higher concentrations (2.0 and 4.0% after 120 min). However, in solutions exposed to UV-C, a drastic reduction in the number of viable trophozoites was observed ( Figure 4). The explanation for this reduction is a limitation for the present study. Although the formation of different transient forms of chlorine has been demonstrated, including Cl 2 , Cl À 3 , HClO À , ClO À and Cl À , during the exposure of highly concentrated NaCl solutions to gamma radiation, the same was not confirmed during exposure to UV-C radiation (Büppelmann et al. ; Paviet-Hartmann et al. ).
We hypothesize that the exposure of trophozoites to NaCl solutions makes them more sensitive to the biocidal effect of UV-C. Further studies are desirable to elucidate the physicochemical phenomena that occur when aqueous solutions of NaCl are exposed to UV-C and its implication in the viability of microorganisms.
The data show that the cysts were more resistant to the effect of NaOCl and NaCl photolysis by UV-C compared with trophozoites. The resistance of the A. castellanii cysts ( Figures 5 and 7) is attributed to its double wall consisting of cellulose and other biomolecules in both layers (Garajová et al. ). The wall of the cysts is essentially opaque to radiation, which prevents the direct damage of intracellular biomolecules by radiation energy or by the intracellular formation of reactive oxygen species, as shown for E. coli    Figures 6 and 8).
The results (Figures 5 and 7) are in agreement with the findings of other researchers (Zhou et al. ) and suggest that A. castellanii cysts are more resistant than C. parvum oocysts.

Practical implications
Our data strongly suggest that the implementation of FAC photolysis through the use of UV-C lamps in drinking water treatment processes will ensure the inactivation of chlorine-resistant microorganisms, such as A. castellanii Inactivation of cysts by NaCl photolysis in the water matrix is a promising way to eliminate chlorine-resistant microorganisms, for example Acanthamoeba spp., Naegleria spp. and Cryptosporidium spp., whose presence in swimming pool water represents a health risk (Zhou et al. ; Paknejad et al. ). This method is particularly desirable because it is inexpensive, as in addition to NaCl being relatively less expensive than other disinfectants such as chlorine, its use does not require the continued addition of new doses after each photolysis disinfection session. In addition, the only presence of NaCl in water exerts a biocidal influence on microorganisms, including to those that are chlorine-resistant, as shown to Naegleria fowleri (Lam et al.

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, which is the etiologic agent of primary amoebic encephalitis, which is a disease of high mortality (Chen et al. ).
Our data also suggest that the photolysis of FAC and NaCl can also be applied in the treatment of wastewater,

CONCLUSIONS
In this study, we evaluated the effect of NaCl or NaOCl photolysis by UV-C light on the viability of trophozoites and A.
castellanii cysts in the water matrix. Our results showed that this process is capable of inactivating A. castellani trophozoites and cysts. The inactivation of A. castellanii was more favored by the time of exposure to UV-C than by the concentration of NaCl or NaOCl.