Research Article: Efficiency of chlorine and UV in the inactivation of Cryptosporidium and Giardia in wastewater

Date Published: May 13, 2019

Publisher: Public Library of Science

Author(s): Folasade Esther Adeyemo, Gulshan Singh, Poovendhree Reddy, Faizal Bux, Thor Axel Stenström, Adelaide Almeida.


Wastewater from different sources is contaminated by protozoan parasites including Cryptosporidium and Giardia. Many protozoan parasites are becoming resistant to chemical treatment. The challenge of finding alternatives is presented to researchers by exploring other methods of eliminating protozoan parasites from wastewater. The aim of this study was to assess the speciation and the viability of Cryptosporidium and Giardia in environmental samples with the specific objective of evaluating if effluent chlorination and UV affect the viability. Different doses of chlorine with different exposure times were experimented with both distilled water and waste water spiked with (oo)cysts derived from environmental samples. UV irradiation at different doses was also experimented using the same spiked samples. Two methods of quantification and detection, namely, microscopy and flow cytometry, were used in the experiment. Two vital dyes, Syto-9+PI and DAPI+PI, were the used for staining the collected wastewater samples. It was found that the (oo)cysts responded to chlorination and UV treatments with Giardia responding better than Cryptosporidium. Giardia responded very well to UV irradiations with almost 0 percent remaining viable after a low dose of UV. Cryptosporidium was found to be resistant to chlorination even at high doses but responded well to high UV doses. DAPI+PI dye gave a lower mean percentage viability values than Syto-9+PI. Flow cytometry gave higher mean percentage than microscopy from the results. It is concluded that UV is a promising alternative to Chlorine in removing Cryptosporidium and Giardia from waste water. Appropriate treatment method for wastewater is necessary to minimize water resources pollution when wastewater is released into water systems.

Partial Text

There is a great concern for the safety and security of environmental water which invariably affects the quality of drinking water and aquatic habitats. Water may be contaminated by environmental pollution and industrial activities. Environmental pollution may result from land use around water sources especially along informal settlements of developing countries. Some small-scale agricultural activities around water sources may also introduce contaminants in the runoff flowing into rivers, ponds and lakes. Wastewater from wastewater treatment plants may be contaminated with many microorganisms including Cryptosporidium and Giardia [1]. Many of these organisms are difficult to remove which is a challenge for effective wastewater treatment. When most contaminants are removed, Cryptosporidium and Giardia may be left because of their resistance to many treatment chemicals and methods [2]. Relatedly, waterborne outbreaks are always traced to many water treatment plants even after they might have met all microbiological and chemical standards for discharge [3].

In this study, different doses of chlorine and varying exposure times were used with distilled water and wastewater spiked with (oo)cysts from wastewater samples. In parallel, same samples were treated with UV irradiation treatment at different UV doses. Microscopy and flow cytometry were used for the detection of Cryptosporidium and Giardia in wastewater. Syto-9+PI and DAPI+PI dyes were used for staining the collected wastewater samples and results were compared.

Ten-microliter aliquots of oocyst suspension was viewed under epifluorescence microscope equipped with a UV filter block (365-nm excitation, 445-nm emission) for DAPI and a green filter block (500-nm excitation, 630-nm emission) for PI. Images were captured using a Zeiss AxioCam MRc (Carl Zeiss, Germany) camera and image quantification was carried out using the Zeiss AxioVision Release 4.6 (12–2006) imaging software. Proportions of ruptured (ghost), PI-positive (PI+), DAPI positive/PI positive (DAPI+PI+), DAPI-positive/PI-negative (DAPI+ PI-), DAPI negative PI-negative (DAPI- PI-) oocysts were quantified in each sample.

The flow cytometry analysis was performed on FACSCalibur BD Biosciences, Sydney standard model, with three PMTs equipped with standard filters (FL1: LP 695/40 nm; FL2: LP 585/42 nm; FL3: LP 488/10 nm; FL4: LP 780/60 nm; FL5: LP 616/23 nm FL6: LP 530/30 nm). The machine was fitted with an argon ion laser operating at 488 nm Argon Laser and with cell Quest Pro software (version 4.0.2, BD Biosciences, Sydney). Acquisition settings were defined using a no stained sample (autofluorescence), adjusting the PMTs voltage to the first logarithmic (log) decade. Instrument controls followed standard procedures adapted from [31]. Sheath fluid consisted of 2.0 mM potassium phosphate buffer (pH 6.8). The detectors used were forward angle light scatter (FALS) and side-angle light scatter (SALS).

Effects of UV on viability at different doses are presented and discussed in section (a) while section (b) presents the effect of different doses of chlorine and duration of exposure on viability. In both sections, comparison between UV and chlorination are presented and discussed. In section (c), effect of turbidity on viability and comparison between the results obtained using the two different dyes, Syto-9+PI and DAPI+PI, are presented and discussed. Finally, Section (d) presents the comparison between microscopy and flow cytometry results during the experiment.

This study presented the reactions of Cryptosporidium oocysts and Giardia cysts to UV irradiations and chlorine at different doses and exposure time. It has been found that effect of UV on Giardia cyst is more pronounced than the effect of chlorine at 0.5 ppm, 2 ppm and 5 ppm. More Giardia cysts became non-viable at lower chlorine concentration compared to Cryptosporidium oocysts. It takes a longer exposure time for Cryptosporidium oocysts reduction in viability. Increasing the chlorine doses resulted in the reduction of viability of Cryptosporidium and Giardia. This same trend was evident with UV irradiations. However, longer exposure time can also play significant role in the reduction in viability of Cryptosporidium and Giardia in wastewater treatment notably up to a threshold exposure time. At that threshold exposure time, further exposure has little or no effect on the viability. It was concluded in the study that Cryptosporidium oocyst is more resistant to both chlorine and UV irradiations than Giardia in wastewater treatment. The study suggests that Cryptosporidium may be resistant to chlorine treatment. Combined treatment of UV and chlorine may be needed to eradicate Cryptosporidium in water treatment in a shorter duration.