Efficiency of chlorine and UV in the inactivation of Cryptosporidium and Giardia in wastewater (2024)

Purification of oocysts

Isolation and purification of oocysts were done from fresh raw wastewater effluent using the glucose gradient centrifugation technique and flotation in glucose-NaCl solution [19]. Samples were filtered through metal mesh-sieves 100 μm to remove debris and large particles, and later centrifuged at 3000 x g for 10 min. The sediments were suspended in PBS-Tween 80. The specific gravity of the flotation fluid was 1.07 g/mL. 300 mL of the flotation fluid was prepared by adding 50 g of glucose to 100 mL saturated NaCl solution and mixing thoroughly. 150 mL of purified water (such as Milli-Q or Super-Q) was added and mixed thoroughly until the solution was hom*ogenous and transparent. Flotation fluid was carefully added by inserting a borosilicate glass Pasteur pipette into the same tube containing the sample so that the tip rests against the bottom of the tube. This would allow it to rise from below with a transparent layer with the sample material (mixed with PBS-Tween 80) above it. The tubes were centrifuged at 750 rpm for 10 min. Then supernatant above the layer of flotation fluid and pellet was transferred into a new tube. After that, the sample was washed by filling the tube up with purified water and then centrifuged at 3000 rpm for 10 minutes. The supernatant was aspirated down to 5 mL. The samples were then washed for two more times (3 x 10 min centrifugation in total) or more depending on how dirty the sample was. The final volume was 5 mL of purified (oo)cysts suspension that was left in the tube which was then vortexed and stored in the fridge at 4°C for future use.

Chlorine treatment of (oo)cysts

Spiked solutions were prepared with wastewater and distilled water. The distilled water represents the control for the experiment. The water samples (both distilled water and wastewater) were chlorinated using HTH (High test hypochlorite 65% chlorine) at concentrations of 0.5 ppm, 2 ppm and 5 ppm. The chlorine and UV experiments were performed according to the methods described by Wu, Fang [20] with modifications. In a 50 mL polyethylene bottles containing the chlorinated distilled water (control) or wastewater, a portion (1 mL) of purified (oo)cysts suspension, approximately 74,600 oocysts per mL were added to each bottle and kept in the dark for adsorption at varying time intervals (15 mins, 30 mins, 60 mins and 120 mins). The samples were collected at different time intervals, and the chlorine reaction was quenched with sodium thiosulphate (Na₂S₂O₃) at concentration ratio of Na₂S₂O₃ to chlorine of 4:1 [21, 22]. The same amount of (oo)cysts spiked in chlorinated distilled water and wastewater was added to the controls test (untreated), which was incubated and processed the same way as the chlorine treated bottles were. This was aimed at determining the concentration of (oo)cysts that may be present in the sample (distilled water and wastewater) before spiking, that might contribute to an increase in concentration of the spiked samples, most especially in the wastewater sample. The untreated control was also run to determine the level of permeability shown by the (oo)cysts to the stains. This was to confirm that each stain is permeable to what it was supposed to stain. The pH and total dissolved solid of the spiked samples (distilled water and wastewater) were measured after filtration and before spiking with the respective (oo)cysts. The pH was 7.6 and 6.8 respectively. The total dissolved solid (TDS) was 35.5 mg/L and 250 mg/L. The wastewater used was clear to naked eye though its turbidity was not measured.

Distilled water and wastewater samples, at different chlorine concentrations (0.5 ppm, 1 ppm, 2 ppm, 5 ppm) were spiked with (oo)cysts suspension and tested at various time interval (15, 30, 60, and 120 min) for chlorine residual. Iodometric method was used for the residual chlorine test since the lowest concentration in this analysis is 0.5 ppm. However, the starch-iodide titration was used to monitor the concentration level of the chlorine during the reaction time. This was achieved by titrating sodium thiosulphate (Na₂S₂O₃) against the residual chlorine concentration in the sample. The volume of Na₂S₂O₃ consumed was used to calculate the concentration of chlorine in the sample with Eq (1).

For the UV experiments, both samples (distilled water and wastewater) were exposed to UV irradiation. A portion (1 mL) of purified (oo)cysts suspension, approximately 74,600 oocysts per mL was added to each bottle containing distilled water and wastewater. The experimental bottles used were 50 mL polyethylene bottles, round and transparent [23] with the height and diameter of 91 mm and 32 mm respectively. The surface area exposed to UV was 804 mm². The experimental bottles were placed in a medium pressure mercury lamp (Heraeus GPH 212T5L/4, 10 W) and the temperature was maintained in a controlled water bath (model: D-91126, Schwabach FRG, Germany) at 25(±0.2)^oC. The samples were collected at different time intervals of 10 seconds, 20 seconds, 40 seconds, 80 seconds and 160 seconds corresponding to the UV doses of 5.2mJ/cm², 10.4 mJ/cm², 20.8 mJ/cm², 41.6 mJ/cm² and 83.2 mJ/cm² respectively. Control sample bottles were kept without UV application, incubated and processed the same way as the UV treated bottles were. The viability reduction was evaluated according to Pereira, Costa [24] by comparison between the viability of the oocysts submitted to disinfectants and oocysts control, considered 100% viable. UV doses were measured in millijoules seconds per cm² (mJ/cm²) and were calculated using the following parameters. The relationship between these parameters can be described by the following simplified equation:

Where, I = Intensity measured in milliwatts per cm² (mW/cm²), UVT = UVTransmittance, T = Exposure time (t) (seconds)

UV dose is the product of UV light intensity and time. Dose is sometimes referred to as fluence. DOSE is equal to Intensity x Time = millijoules/(sec)(cm²) x time = mJ/cm². Therefore, the UV fluence was calculated as in Eq (3) [25–27].

Viability test

The viability of the (oo)cyst after treatment with chlorine and UV was assessed and determined using vital dye Syto-9+Propidium iodide, and with the inclusion of DAPI+ Propidium iodide staining. Stained (oo)cysts were counted with flow cytometer and epifluorescence microscope. The nucleic acid dyes were supplied by Molecular Probes (Eugene, OR, U.S.A.). Live/Dead viability kit (L-34856) for flow cytometry and Live/Dead viability kit (L-7012) for microscopy and quantification assays were used. Two live/dead double-staining viability kits were considered.

Staining procedure with Syto-9 and PI

The Live/Dead BacLight Viability assay utilizes mixtures of Syto-9 green fluorescent nucleic acid stain and the red fluorescent nucleic acid stain, propidium iodide. According to explanation given by [28], the two stains vary both in their characteristics and in their ability to enter healthy bacterial cells. The Syto-9 stain labels bacteria with both intact and damaged membranes when used alone. Whereas, propidium iodide permeates only bacteria with damaged membranes, competing with the Syto-9 stain for nucleic acid binding sites when both dyes are present. When mixed in recommended proportions, Syto-9 stain and propidium iodide produce green fluorescent staining of bacteria with intact cell membranes and red fluorescent staining of bacteria with damaged membranes [28].

Viability of the (oo)cysts were determined by double-staining of (oo)cysts with the use of Live/Dead BacLight kit (Invitrogen) was performed according to [29] adapted from [30] with little modification. The Live/Dead BacLight kit was removed from -20°C light protected storage and allowed to thaw at room temperature. Equal volumes of the two reagents (Syto-9 and propidium iodide) were combined in a 1.5 mL Eppendorf tube and mixed. The tube was wrapped with aluminium foil in order to avoid light penetration into the tube containing the staining solution. Three microliters of this mixture were then added to 97μL of DW, containing 2 × 10⁴ oocysts. After incubation at 37°C for 60 min, 10 μL of aliquots were placed on a slide and microscopical observations were made. Samples were observed under Axiolab Zeiss fluorescence microscope, using either 400× or 1000× magnification. Colour and intensity of live (dark green) and dead (bright red or orange/yellow) (oo)cysts staining were visually assessed. Another aliquot from staining solution 100 μL was in the fluidic sample tube and analysed with flow cytometer. A green filter block was used to examine the Syto-9 and PI (propidium iodide) stained (oo)cysts. Nonviable (oo)cysts were fluorescent red, while viable (oo)cysts were fluorescent green.

Reagent quality and performance check of the fluorogenic dyes

Method performance of the dyes were checked to confirm if the specific dye was staining what they were meant to stain and penetrate into. This was to assess the usefulness of these dyes as indicators of viability for Cryptosporidium oocysts and Giardia cysts. These stains vary both in their spectral characteristics and in their ability to penetrate into the cells. Therefore, samples were also analysed separately for each stain (DAPI only, PI only and Syto-9 ONLY) along with all samples analysed with DAPI+PI and Syto-9 + PI. This was performed to compare and to establish performance criteria of each stain in order to determine if the results of analyses from each stain was correct.

Staining procedure DAPI and PI

Incubation of oocysts with DAPI and PI was performed according to the protocols described by [15]. Working solutions of DAPI (2 mg/mL in absolute methanol) and PI (1 mg/mL in 0.1 M PBS, pH 7.2) were prepared and stored at 4°C in the dark. 100 μl of test suspension containing approximately 60.1 X 10³ to 74.6 X 10³ were incubated simultaneously with 10 μl of DAPI working solution and 10 μl of PI working solution at 37°C for 2h. (oo)cysts were viewed by fluorescence microscopy and also analysed by flow cytometry.

Data analysis

Percentages of Cryptosporidium and Giardia viabilities were calculated for the UV and chlorine exposures at different exposure times and doses for all the trials. The mean and standard deviation of percentage viability of Cryptosporidium and Giardia after exposure to UV radiation and chlorine were also calculated and reported. In this study, descriptive analysis was done on the percentage viability to determine the mean and standard deviation of Cryptosporidium and Giardia in distilled water and wastewater. This was to assess the relationship between the mean percentage viability between the different doses of chlorine and UV on both distilled water and wastewater. The two dyes were compared graphically. This was to determine the effect of different dyes on the results of mean percentage viability. The relationship between the methods of detection, viz cytometry and microscopy, were also compared with the use of graphs.

Effect of UV on viability

Table 1 presents the effect of the UV dose on the die-off of Giardia cysts and Cryptosporidium oocysts as measured with microscopic counts using DAPI+PI and Syto-9+PI. Table 2 presents the same effect as measured with flow cytometry counts using same dyes (DAPI+PI and Syto-9+PI). The comparative impacts on the die-off in distilled water versus in wastewater are further presented in Fig 1 and the effect due to different stains in Fig 2. Overall, the UV efficiency is higher in Giardia than in Cryptosporidium but with a common cut-off value of 20 mJ/cm² based on selected doses (Table 1). [32] reported that UV irradiation usually inactivates Cryptosporidium by attacking the nucleic acid and thereby preventing multiplication of the parasite. Giardia is thus less resistant to UV irradiations than Cryptosporidium in line with the conclusion by [33] that Giardia responded very well to UV irradiation. It can be observed that UV irradiation has more effect on Giardia than chlorine (Table 1). This is due to the killing effect exhibited by UV on Giardia because of its effect on its DNA [33]. The same effect relates to Giardia destruction [33]. Die-off of the (oo)cysts is directly proportional to the increase in exposure time and the UV doses [34]. The sensitivity of Giardia to UV irradiation was higher than Cryptosporidium as found in the obtained results and was also observed by [33]. As a support to the obtained results in the present study, Campbell and Wallis [35] showed that UV irradiation at 20 to 40 mJ/cm² is capable of killing 99.9% of Giardia cysts. Rahdar and Daylami [33] further proved that UV radiation of 50mJ/cm² at 280nm can destroy only Giardia cysts from feaces of infected patients to a maximum of 75%. However, the results in this present study are slightly different from this. The test regime of this study, where (oo)cysts were extracted from environmental water samples, was different from those studies [33, 35]. This may be accountable for the different results generated in this present experiment.

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Fig 1

Mean percentage viability of Cryptosporidium and Giardia related to UV doses in treatment with spiked samples in distilled water and wastewater samples.

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Fig 2

Mean percentage viability of Cryptosporidium and Giardia at different UV doses using Syto-9+PI and DAPI+PI dyes.

It was reported by [36] that Cryptosporidium 2 log excystation reduction requires high UV dose of 230mWs/cm². This was the recommended dose for the purified water with fresh faecal samples used for their experiment. They further explained that the UV dose required for 1-, 2-, and 4-log reduction in infectivity in the purified water with fresh faecal samples were 0.48, 0.97 and 1.92 mWs/cm² respectively which are higher than the doses used in the present study. Moreover, a 200 times higher dose will be required for a 2-log reduction in viability than reduction in infectivity as assessed by in vitro excystation. This demonstrates that if Cryptosporidium oocysts are exposed to low dose of UV irradiation, they may not be infectious but they will still be able to excyst in the purified water with faecal samples [36].

In Table 3, the mean percentages and standard deviation of viable Cryptosporidium and Giardia at different UV doses gave a remaining 1±1% of viable Cryptosporidium at 83.2 mJ/m². At 20.8 mJ/m², all Giardia were already dead. This further confirms that higher UV doses are required to eliminate Cryptosporidium than Giardia. Prolonged UV exposure could also be more potent than short exposure as reported by [34]. Moreover, at low UV doses, a fraction of viable Cryptosporidium and Giardia are affected as found in Table 1, where a remaining 47% and 25% of Cryptosporidium and Giardia were counted as viable respectively at 5.2 mJ/cm² dose. It can further be established that UV irradiations have more effect on Giardia than Cryptosporidium in all the trials as reported (Table 1; Figs ​Figs11 and ​and2).2). Cryptosporidium reduction in viability due to UV exposure in wastewater is encouraging and also in agreement with [37] who found that solar UV can rapidly inactivate Cryptosporidium in environmental waters.

Fig 1 illustrates the reduction in mean percentage viability of Cryptosporidium and Giardia as related to treatment with different UV doses. The results presented in Fig 2 represent the mean percent viability using both dyes for the 5 trials (that is Syto-9+PI in trials 1 to 3 and DAPI+PI in trials 4 to 5). All Giardia originally present were eliminated at 20.8 mJ/cm² of UV dose which confer its efficiency in removing Giardia from wastewater. Less than 2% of Cryptosporidium was viable after 83 mJ/cm² UV exposure. An increase in exposure dose can significantly reduce the viability of both Cryptosporidium and Giardia in water. It was found in this study that UV disinfection was much more effective than chlorination. This same conclusion was noted by [38].

Effect of chlorination on viability

Table 4 presents the mean percentages of viable Cryptosporidium and Giardia after exposure to 0.5 ppm, 2 ppm and 5 ppm of chlorine during different exposure times. For 0.5 ppm, Giardia became non-viable after 60 minutes of exposure except in trial 4 where they are non-viable after 30 minutes. Half of the Cryptosporidium were still viable (42 to 47% for distilled water and 42 to 51% for wastewater) after 120 minutes exposure time. The findings of [33] supports this finding that lower concentration of chlorine results in the elimination of small amount of oocysts. Since the reduction in distilled water and in wastewater were similar, the data has been treated jointly. Thus, the mean viable fraction of Cryptosporidium decreased from 86±2% after 15 minutes’ exposure to 0.5 ppm of chlorine to 46±4% viability after 120 minutes. However, 0.5 ppm of chlorine was not judged as effective for Cryptosporidium removal even after long exposure time (120 minutes) when compared to the effect on Giardia in wastewater (Fig 3). A low dose of chlorine (0.5 ppm) with about 60 minutes exposure time may be sufficient for Giardia removal as all Giardia present were non-viable after 60 minutes. These results were corroborated in the World Health Organisation study [39] that reported that Giardia is less resistant to disinfection including chlorination. In Table 4, at 2 ppm, it was found that all Giardia became destroyed after 15 minutes in all the 4 trials (2 to 5) except in trial 1 where Giardia became destroyed after 30 minutes. It can be found that the same number of Giardia was destroyed with chlorine dose of 0.5 ppm (60 minutes exposure time) and 2 ppm (15 minutes exposure time). The effectiveness can thus be either enhanced by increased exposure time or through increase in dose [40].

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Fig 3

Mean percentage viability of Cryptosporidium and Giardia after prolonged exposure to 0.5 ppm, 2 ppm and 5 ppm of chlorine treatments for distilled water and wastewater.

Cryptosporidium is smaller and more robust than Giardia which makes it to withstand the chlorine doses that are used in the water treatment processes. Their resistance has caused several water borne outbreaks worldwide [7, 41–43]. Water authorities need to combine multiple treatment barriers for efficient Cryptosporidium removal in wastewater [16].

The results presented in Fig 4 compares the mean percentage viability using direct microscopy with Syto-9+PI in trials 1 to 3 and DAPI+PI in trials 4 to 5. In Fig 4(C), there is a steady reduction in viability of Giardia until after 30 minutes when all Giardia were dead for 5 ppm. Therefore, increasing the duration of exposure may not really influence the viability of Giardia as found after 50 minutes for both 0.5 ppm and 2 ppm (Figs 4(A) and 4(B)) and 30 minutes for 5 ppm (Fig 4(C)). For Cryptosporidium, a slight reduction in viability was invariably noticed after 100 minutes for 5 ppm (Fig 4(C)) and after 50 minutes for 0.5 ppm and 2 ppm (Fig 4(B) and 4(C)). In conclusion, an increase in exposure time may be significant up to a threshold, in this case, at 100 minutes for 5 ppm and 50 minutes for 0.5 ppm and 2 ppm, after which increase in exposure time may not have any appreciable reduction in viability. There were no significant trends as depicted by Fig 4. This further confirms that increasing chlorine dose from 0.5 ppm to 2 ppm may not have effect on the viability of Cryptosporidium unlike an appreciable reduction with the viability of Giardia. This points out that Cryptosporidium is more resistant to chlorine than Giardia when exposed under the same environmental conditions [16, 44, 45].

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Fig 4

Mean percentage viability of Cryptosporidium and Giardia at different chlorine exposures using Syto-9+PI and DAPI+PI dyes for distilled water and wastewater (microscopy method).

All Giardia present were eliminated after 15 minutes exposure time for 5 ppm which is similar to 2 ppm dose (Table 4). Increasing chlorine dose from 2 ppm to 5 ppm, makes no difference to Giardia viability. It shows that it is a waste of resources to increase chlorine dose from 2 ppm to 5 ppm for the sole aim of Giardia removal in wastewater. There was appreciable viability reduction for Cryptosporidium with chlorine dose increase from 2 ppm to 5 ppm but no reduction in Giardia for same increase (Table 4). More than half of Cryptosporidium was destroyed after 15 minutes exposure with 5 ppm (42±8% viability). Similarly, after 120 minutes of exposure with 5 ppm dose, almost 90% of Cryptosporidium was eliminated (15±3% viability). This shows that increasing the chlorine dose to 5 ppm has significant effect on the reduction in viability of Cryptosporidium with almost total elimination. However, chlorine may not remove all Cryptosporidium present in wastewater as there was no significant reduction in viability after 100 minutes of exposure. Fig 3(C) shows that mean percentage viability is inversely proportional to duration of exposure with a sharp decrease in mean percentage viability from 15seconds to 80 minutes and slight decrease from 100 minutes to 120 minutes showing that an increase in exposure duration has limited effect at 100 minutes to 120 minutes and beyond. Additionally, it can be inferred that both duration of exposure and dose have effect on viability for both Cryptosporidium and Giardia but at different stages [44]. The effect of increase in dosage is more pronounced at the beginning of experiment where there are sharp decreases in viability.

At a low dose, duration of exposure may need to be increased for a reduction in viability. This will ensure that more Cryptosporidium and Giardia are eliminated over a longer time with slow reaction with chlorine. In summary, an increased dose is more effective than exposure duration. This assertion can be supported with findings in Table 4 which presents the mean and standard deviation of viability of Cryptosporidium and Giardia after exposure to chlorine. At 15 minutes, about 86±2%, 80±6% and 42±8% of Cryptosporidium was still viable at 0.5 ppm, 2 and 5 ppm respectively. The comparative values at 120 minutes were 46±4%, 50±6% and 15±3% of Cryptosporidium were viable showing the effect of duration of exposure and increase in chlorine dose. The same phenomenon was observed for Giardia where increase in dose from 0.5 ppm to 2 ppm reduced viability from 57±32% to 7±0% at 15 minutes. However, further increment from 2 ppm to 5 ppm did not decrease viability as it remained at 7±0%. Increasing the duration of exposure from 15 minutes to 30 minutes has a pronounced effect on to viability as it decreased to 30±21% from 57±32% for 0.5 ppm and 0% from 7±0% for 2 ppm and 5 ppm. Further increase in duration of exposure from 30 minutes to 60 minutes and later to 120 minutes did not decrease viability of Giardia as all Giardia present were non-viable after 30 minutes (Table 5).

The same trend was observed for UV exposure in Table 3. After 10.4mJ/cm², all Giardia present became dead. Further increase in dose became unnecessary after 10.4 mJ/cm². Conversely, there was a slight decrease in the viability of Cryptosporidium after 20.8 mJ/cm² from 5±0% to 3±1% and later to 1±1% after 20.8 mJ/cm², 41.6 mJ/cm² and 83.2 mJ/cm² respectively unlike sharp decrease experienced after 5.2 mJ/cm² (100% to 47±3%), 10.4 mJ/cm² (4.7±3% to 22±1%) and 20.8mJ/cm² (22±1% to 5±0%). In spite of this, Table 5 shows that UV is more effective in removing Cryptosporidium and Giardia in wastewater than chlorine even at higher doses of chlorine. While chlorine at 5 ppm has 15±3% viability even at 120 minutes, UV has 3±1% viability at 41.6 mJ/cm²(80 seconds). This agrees with previous studies where efficacy of UV over chlorine has been demonstrated in the removal of Cryptosporidium and Giardia in wastewater [9, 36, 37, 46]. Chlorine at higher doses may not be recommended due to the toxic by-products such as trihalomethanes (THMs) [47]. Higher doses above 2 ppm concentrations are not used in conventional water treatment and also not economically viable in relation to water treatment stations [24].

Effect of turbidity

When comparing the results between the distilled water and the wastewater experiment, a slightly higher fraction of viable (oo)cysts were obtained in the wastewater samples especially in chlorine treatment (Tables ​(Tables33 and ​and4).4). The turbidity would affect the water treatment efficiency using UV irradiation as depicted in Table 3 when both Cryptosporidium and Giardia are considered together. The mean percentage viabilities for distilled water and wastewater for all UV exposures were comparable for Cryptosporidium. This shows that viability was not really affected by opacity for Cryptosporidium removal. For Giardia, higher values were recorded for wastewater in many of the trials showing that higher turbidity causes increase in viability due to UV opacity. The mean percentage viability for distilled water (Trials 1 and 2) and wastewater (Trials 3 to 5) in Table 4 are however comparable with values for wastewater slightly higher at lower exposure durations (15 to 30 minutes) especially for Giardia. Turbidity, thus, slightly affected the treatment efficiency, especially when low doses of chlorine were applied. Cryptosporidium percentage viability for distilled water and wastewater were comparable. Water with low turbidity responds better to chlorine treatment than turbid water (wastewater).

Comparison between microscopy and flow cytometry

When assessing the results obtained using microscopy as compared to flow cytometry for UV irradiations in Table 5, the results for both methods showed decreasing viability with increasing irradiation where microscopy gave slightly higher viability. It was also evident that percentages of viability were presented for both microscopy and flow cytometer (Table 6). It can be observed further that microscopy has higher mean percentage viability value at 15 minutes while flow cytometry has higher percentage values at 30 minutes, 60 minutes and 120 minutes for 0.5 ppm. This shows that flow cytometry gives higher mean percentage viability values than microscopy especially at 0.5 ppm chlorination for both distilled water and wastewater. Tables ​Tables44 and ​and55 at Trials 2 and 3 shows that Cryptosporidium and Giardia have higher mean percentage viability values at Trial 2 (distilled water) than Trial 3 (wastewater) using the same dye (Syto-9+PI) for staining. This shows that under the same condition and staining, chlorination may have lower effect on wastewater than distilled water. In this experiment, turbidity did not have high significance on the treatment efficiency in all the trials. This may be due to the type of stains used.

Samples of flow cytometry histograms and dot plots showing viability of Cryptosporidium and Giardia are given in Figs ​Figs55 and ​and6.6. Fig 5 depicts exposure to UV irradiation after 20.8mJ/cm² while Fig 6 presents exposure to 5 ppm of chlorine after 15 minutes. Other similar flow cytometry histograms and dot plots showing viability of Cryptosporidium and Giardia at different doses of UV and chlorine at different exposure times are available in the supporting document. In the supporting document, Figs A–E in S1 File present the diagrams for UV viability while Figs F to I in S1 File, Figs J–M in S1 File, and Figs N- Q in S1 File present the diagrams for 0.5 ppm, 2 ppm, and 5 ppm of chlorine respectively. Samples of Region P1 and P2 represent gates for non-viable and viable (oo)cysts with P1 as side-scatter (SSC) against forward side-scatter (FSC). Histograms represent percentages of non-viable (oo)cysts plotted with count against PE-CF594-A and PerCP0-Cy5-5-A. The gates P1 and P2 were used to illustrate a subgroup of data. Statistics was generated using the population and also to limit the number of events collected. The gated region shows the sample of mixed population of cells. The results of all the cytometry histogram and dot plots are presented in Table 6 for chlorine and Table 5 for UV.

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Fig 5

Flow cytometry histogram and dot plots showing viability of Cryptosporidium and Giardia at 40 seconds after exposure to UV irradiation.

Note: Samples of Region P1 and P2 represent gates for non-viable and viable (oo)cysts with P1 as side-scatter (SSC) against forward side-scatter (FSC).

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Fig 6

Flow cytometry histogram and dot plots showing viability of Cryptosporidium and Giardia at 15 seconds after exposure to 5 ppm of chlorine.

Note: Samples of Region P1 and P2 represent gates for non-viable and viable (oo)cysts with P1 as side-scatter (SSC) against forward side-scatter (FSC).

Comparison between Syto-9+PI and DAPI+PI vital dyes

Tables ​Tables3,3, ​,4,4, ​,55 and ​and66 present the comparison between the results of microscopy and flow cytometer. While the first 3 trials present the results using Syto-9+PI stains, the last 2 trials present results using DAPI+PI stains. The type of stain used for the experiment can have effect on the results as can be found in Fig 2. The figure presents the results of the two dyes differently. Syto-9+PI was used for trials 1 to 3 and DAPI+PI was used for trials 4 to 5. Syto-9+PI dyes gives slightly higher percentages than DAPI+PI. Therefore, Syto-9+PI is slightly more sensitive than DAPI+PI for Cryptosporidium and Giardia. This is notable because vital dyes may overestimate Cryptosporidium and Giardia in wastewater [48].

Largely, the results of mean percentage viability values using Syto-9+PI stains (trials 1 to 3) for (oo)cysts in Table 6 and Fig 4 were slightly higher than those using DAPI+PI stains (trials 4 to 5) at the initial stages of the experiment (Trials 1 to 3) and became comparable at Trials 4 and 5 because of overestimation of Cryptosporidium and Giardia as mentioned earlier [48]. The results of percentages of flow DAPI and PI of (oo)cysts were presented for different doses and exposure time for chlorine (Table 6). At 0.5 ppm, 85±3% were viable at 15 minutes and 17% were dead. This increased to 66±3% viable and 34% dead at the end of the trials at 120 minutes. The same results were recorded at 2 ppm. Nonetheless, at 5 ppm, 41±7% was viable and 59% dead at 15 minutes and 12±0% viable and 88% dead at the end of the trial (120 minutes). In Table 5, the efficacy of UV was amply demonstrated in the trials that only 3% were viable at 83.2 mJ/cm² (160 minutes) and 97% dead. At 10.4 mJ/cm²(20 seconds), 11.0% were alive, which is lower than the highest dose of 5 ppm of chlorine (41%) at 15 minutes. Meanwhile after 20 seconds, there was a slight reduction in percentage alive with UV exposure unlike a significant decrement in percentage from 0 to 10seconds and 10 seconds to 20 seconds which was 56% and 88% respectively. Surprisingly, increase in the duration afterwards was not significant. [36] suggested oocysts has high resistance to UV irradiation and high UV dose may be required. Then again, UV may not remove all incidences of (oo)cysts as 3% was still alive after 83.2 mJ/cm² (160 seconds).

S1 File

Flow cytometry histogram and dot plots showing viability of Cryptosporidium and Giardia at different durations after exposure to UV irradiation and different doses of chlorine.

Fig A. Flow cytometry histogram and dot plots showing viability of Cryptosporidium and Giardia at 10 seconds after exposure to UV irradiation. Fig B. Flow cytometry histogram and dot plots showing viability of Cryptosporidium and Giardia at 20 seconds after exposure to UV irradiation. Fig C. Flow cytometry histogram and dot plots showing viability of Cryptosporidium and Giardia at 40 seconds after exposure to UV irradiation. Fig D. Flow cytometry histogram and dot plots showing viability of Cryptosporidium and Giardia at 80 seconds after exposure to UV irradiation. Fig E. Flow cytometry histogram and dot plots showing viability of Cryptosporidium and Giardia at 160 seconds after exposure to UV irradiation. Fig F. Flow cytometry histogram and dot plots showing viability of Cryptosporidium and Giardia at 15 minutes after exposure to 0.5 ppm of chlorine. Fig G. Flow cytometry histogram and dot plots showing viability of Cryptosporidium and Giardia at 30 minutes after exposure to 0.5 ppm of chlorine. Fig H. Flow cytometry histogram and dot plots showing viability of Cryptosporidium and Giardia at 60 minutes after exposure to 0.5 ppm of chlorine. Fig I. Flow cytometry histogram and dot plots showing viability of Cryptosporidium and Giardia at 120 minutes after exposure to 0.5 ppm of chlorine. Fig J. Flow cytometry histogram and dot plots showing viability of Cryptosporidium and Giardia at 15 minutes after exposure to 2 ppm of chlorine. Fig K. Flow cytometry histogram and dot plots showing viability of Cryptosporidium and Giardia at 30 minutes after exposure to 2 ppm of chlorine. Fig L. Flow cytometry histogram and dot plots showing viability of Cryptosporidium and Giardia at 60 minutes after exposure to 2 ppm of chlorine. Fig M. Flow cytometry histogram and dot plots showing viability of Cryptosporidium and Giardia at 120 minutes after exposure to 2 ppm of chlorine. Fig N. Flow cytometry histogram and dot plots showing viability of Cryptosporidium and Giardia at 15 minutes after exposure to 5 ppm of chlorine. Fig O. Flow cytometry histogram and dot plots showing viability of Cryptosporidium and Giardia at 30 minutes after exposure to 5 ppm of chlorine. Fig P. Flow cytometry histogram and dot plots showing viability of Cryptosporidium and Giardia at 60 minutes after exposure to 5 ppm of chlorine. Fig Q. Flow cytometry histogram and dot plots showing viability of Cryptosporidium and Giardia at 120 minutes after exposure to 5 ppm of chlorine.

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