Occurrence and variations of cryptosporidium and giardia in wastewater treatment and receiving river basins

Thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy: Health Sciences in the Department of Community Health Studies, Faculty of Health Sciences at Durban University of Technology, Durban, South Africa, 2020.Wastewater disposal may be a source of environmental contamination of Cryptosporidium and Giardia. Releasing untreated wastewater into the environment may result in waterborne or foodborne outbreaks. The presence of Cryptosporidium and Giardia in wastewater is of major concern in human health when wastewater is used for irrigation. The infections influence morbidity with several cases of illnesses including diarrhoea and other gastrointestinal diseases. Mortality is of high incidence, especially among the immunocompromised. Worldwide, Cryptosporidium and Giardia are well-documented in outbreaks from drinking water, recreational water and food. The aim of the study was to demonstrate the level of occurrence of Cryptosporidium and Giardia in selected wastewater treatment plants (WWTP) as well as receiving water bodies and to account for the use of wastewater in irrigation in Ethekwini, South Africa. The occurrence and reduction of Cryptosporidium and Giardia in the WWTPs were investigated, the species determined and their viability assessed. The correlations between E. coli, Enterococci and the concentration of the protozoan parasites were included. Additional specific objectives relate to the effect of effluent chlorination, the seasonal variation and the impact on crops based on the occurrence and quantities of Cryptosporidium and Giardia in the irrigated water. Influent water concentrations reflect the endemicity level in the connected population. This together with water from the intermediate treatment steps and effluent samples in four wastewater treatment plants were investigated monthly (sampled September 2015-April 2016). Conventional filtration and centrifugation methods were used for the detection and enumeration of Cryptosporidium and Giardia. Immuno-magnetic separation (IMS) on concentrated water samples was performed using Dynal anti-Cryptosporidium and anti-Giardia beads and immune-magnetic antibodies and examined with epifluorescence microscopy. In addition, molecular methods were used to differentiate the species of Cryptosporidium and assemblages of Giardia based on polymerase chain reaction (PCR) and quantitative polymerase chain reaction (qPCR). The influent counts for Cryptosporidium oocyst varied between 1.83 to 3.02 log10 oocysts/L, with a corresponding concentration of Giardia cysts ranged from 2.39 to 3.15 log10 cysts/L. The overall mean reduction over the wastewater treatment plants was in the range of 1.35 log10/L to 1.61 log10/L and 1.35 log10/L and 1.62 log10/L for Cryptosporidium and Giardia respectively. Cryptosporidium and Giardia were present in the effluents from all wastewater treatment plants. In general, weak negative to poor positive correlation was found between E. coli, faecal enterococci and the presence of Cryptosporidium and Giardia in all WWTPs. Both Cryptosporidium and Giardia were further detected at the downstream sampling points. The PCR and qPCR speciation did not compare with the traditional specific antibody and microscopic counts. The 18s rRNA gene-based nested PCR resulted in 13 (27%) out of 48 wastewater sample positive for Cryptosporidium. C. parvum was the dominated species followed by C. hominis. In ß-giardin based qPCR, all wastewater samples were positive for Giardia Assemblage A and Giardia Assemblage B. The impact of wastewater on the rivers signifies the possible health risks associated with the use of the effluents for agricultural purposes and the receiving water bodies’ use for domestic and recreational activities. Crops irrigated with the river water from the study area gave concentrations of Cryptosporidium and Giardia in spinach samples of 67 to 480 and 3 to 27 per 100 g vegetable respectively. The contamination of the fresh vegetables analysed mainly arose from the use of the effluent from the WWTP B and the receiving river water used for irrigation. In a pilot study researching the effect of chlorine and UV irradiation, distilled water and wastewater samples spiked with environmentally derived Cryptosporidium oocysts and Giardia cysts which were exposed to different doses of chlorine and UV with different exposure times. Viability quantification and detection was done with microscopy and flow cytometry using two vital dyes, namely Syto9+PI and DAPI+PI. Giardia was affected to a much higher degree than Cryptosporidium, both in relation to chlorination and UV, where in the latter case close to zero percent remained viable even after a low dose. Cryptosporidium was found to be resistant to chlorination but responded well to high UV doses. This is in line with similar earlier investigations done in drinking water. In the comparative assessment between the vital dyes, DAPI+PI dyes gave a lower mean percentage viability value than Syto9+PI; and flow cytometry gave a higher mean percentage value than microscopy. The presence of Cryptosporidium and Giardia in all treated effluents from all investigated wastewater treatment plants exceeded World Health Organisation’s (WHO) risk-based values and South African National Standard’s (SANS:241) limit for raw water and irrigation respectively. Enforced regulations and alternative wastewater treatment methods may ensure a higher level of safety for the environment. Additional information on the occurrence of Cryptosporidium and Giardia in wastewater samples, which is currently lacking or limited in South Africa, will further increase our understanding in formulating risk assessment strategies to map out the level of infection to exposed population

Occurrence and variations of cryptosporidium and giardia in wastewater treatment and receiving river basins

Wastewater disposal may be a source of environmental contamination of Cryptosporidium and Giardia. Releasing untreated wastewater into the environment may result in waterborne or foodborne outbreaks. The presence of Cryptosporidium and Giardia in wastewater is of major concern in human health when wastewater is used for irrigation. The infections influence morbidity with several cases of illnesses including diarrhoea and other gastrointestinal diseases. Mortality is of high incidence, especially among the immunocompromised. Worldwide, Cryptosporidium and Giardia are well-documented in outbreaks from drinking water, recreational water and food. The aim of the study was to demonstrate the level of occurrence of Cryptosporidium and Giardia in selected wastewater treatment plants (WWTP) as well as receiving water bodies and to account for the use of wastewater in irrigation in Ethekwini, South Africa. The occurrence and reduction of Cryptosporidium and Giardia in the WWTPs were investigated, the species determined and their viability assessed. The correlations between E. coli, Enterococci and the concentration of the protozoan parasites were included. Additional specific objectives relate to the effect of effluent chlorination, the seasonal variation and the impact on crops based on the occurrence and quantities of Cryptosporidium and Giardia in the irrigated water. Influent water concentrations reflect the endemicity level in the connected population. This together with water from the intermediate treatment steps and effluent samples in four wastewater treatment plants were investigated monthly (sampled September 2015-April 2016). Conventional filtration and centrifugation methods were used for the detection and enumeration of Cryptosporidium and Giardia. Immuno-magnetic separation (IMS) on concentrated water samples was performed using Dynal anti-Cryptosporidium and anti-Giardia beads and immune-magnetic antibodies and examined with epifluorescence microscopy. In addition, molecular methods were used to differentiate the species of Cryptosporidium and assemblages of Giardia based on polymerase chain reaction (PCR) and quantitative polymerase chain reaction (qPCR). The influent counts for Cryptosporidium oocyst varied between 1.83 to 3.02 log10 oocysts/L, with a corresponding concentration of Giardia cysts ranged from 2.39 to 3.15 log10 cysts/L. The overall mean reduction over the wastewater treatment plants was in the range of 1.35 log10/L to 1.61 log10/L and 1.35 log10/L and 1.62 log10/L for Cryptosporidium and Giardia respectively. Cryptosporidium and Giardia were present in the effluents from all wastewater treatment plants. In general, weak negative to poor positive correlation was found between E. coli, faecal enterococci and the presence of Cryptosporidium and Giardia in all WWTPs. Both Cryptosporidium and Giardia were further detected at the downstream sampling points. The PCR and qPCR speciation did not compare with the traditional specific antibody and microscopic counts. The 18s rRNA gene-based nested PCR resulted in 13 (27%) out of 48 wastewater sample positive for Cryptosporidium. C. parvum was the dominated species followed by C. hominis. In ß-giardin based qPCR, all wastewater samples were positive for Giardia Assemblage A and Giardia Assemblage B. The impact of wastewater on the rivers signifies the possible health risks associated with the use of the effluents for agricultural purposes and the receiving water bodies’ use for domestic and recreational activities. Crops irrigated with the river water from the study area gave concentrations of Cryptosporidium and Giardia in spinach samples of 67 to 480 and 3 to 27 per 100 g vegetable respectively. The contamination of the fresh vegetables analysed mainly arose from the use of the effluent from the WWTP B and the receiving river water used for irrigation. In a pilot study researching the effect of chlorine and UV irradiation, distilled water and wastewater samples spiked with environmentally derived Cryptosporidium oocysts and Giardia cysts which were exposed to different doses of chlorine and UV with different exposure times. Viability quantification and detection was done with microscopy and flow cytometry using two vital dyes, namely Syto9+PI and DAPI+PI. Giardia was affected to a much higher degree than Cryptosporidium, both in relation to chlorination and UV, where in the latter case close to zero percent remained viable even after a low dose. Cryptosporidium was found to be resistant to chlorination but responded well to high UV doses. This is in line with similar earlier investigations done in drinking water. In the comparative assessment between the vital dyes, DAPI+PI dyes gave a lower mean percentage viability value than Syto9+PI; and flow cytometry gave a higher mean percentage value than microscopy. The presence of Cryptosporidium and Giardia in all treated effluents from all investigated wastewater treatment plants exceeded World Health Organisation’s (WHO) risk-based values and South African National Standard’s (SANS:241) limit for raw water and irrigation respectively. Enforced regulations and alternative wastewater treatment methods may ensure a higher level of safety for the environment. Additional information on the occurrence of Cryptosporidium and Giardia in wastewater samples, which is currently lacking or limited in South Africa, will further increase our understanding in formulating risk assessment strategies to map out the level of infection to exposed population</jats:p

Occurrence and variations of cryptosporidium and giardia in wastewater treatment and receiving river basins

Wastewater disposal may be a source of environmental contamination of Cryptosporidium and Giardia. Releasing untreated wastewater into the environment may result in waterborne or foodborne outbreaks. The presence of Cryptosporidium and Giardia in wastewater is of major concern in human health when wastewater is used for irrigation. The infections influence morbidity with several cases of illnesses including diarrhoea and other gastrointestinal diseases. Mortality is of high incidence, especially among the immunocompromised. Worldwide, Cryptosporidium and Giardia are well-documented in outbreaks from drinking water, recreational water and food. The aim of the study was to demonstrate the level of occurrence of Cryptosporidium and Giardia in selected wastewater treatment plants (WWTP) as well as receiving water bodies and to account for the use of wastewater in irrigation in Ethekwini, South Africa. The occurrence and reduction of Cryptosporidium and Giardia in the WWTPs were investigated, the species determined and their viability assessed. The correlations between E. coli, Enterococci and the concentration of the protozoan parasites were included. Additional specific objectives relate to the effect of effluent chlorination, the seasonal variation and the impact on crops based on the occurrence and quantities of Cryptosporidium and Giardia in the irrigated water. Influent water concentrations reflect the endemicity level in the connected population. This together with water from the intermediate treatment steps and effluent samples in four wastewater treatment plants were investigated monthly (sampled September 2015-April 2016). Conventional filtration and centrifugation methods were used for the detection and enumeration of Cryptosporidium and Giardia. Immuno-magnetic separation (IMS) on concentrated water samples was performed using Dynal anti-Cryptosporidium and anti-Giardia beads and immune-magnetic antibodies and examined with epifluorescence microscopy. In addition, molecular methods were used to differentiate the species of Cryptosporidium and assemblages of Giardia based on polymerase chain reaction (PCR) and quantitative polymerase chain reaction (qPCR). The influent counts for Cryptosporidium oocyst varied between 1.83 to 3.02 log10 oocysts/L, with a corresponding concentration of Giardia cysts ranged from 2.39 to 3.15 log10 cysts/L. The overall mean reduction over the wastewater treatment plants was in the range of 1.35 log10/L to 1.61 log10/L and 1.35 log10/L and 1.62 log10/L for Cryptosporidium and Giardia respectively. Cryptosporidium and Giardia were present in the effluents from all wastewater treatment plants. In general, weak negative to poor positive correlation was found between E. coli, faecal enterococci and the presence of Cryptosporidium and Giardia in all WWTPs. Both Cryptosporidium and Giardia were further detected at the downstream sampling points. The PCR and qPCR speciation did not compare with the traditional specific antibody and microscopic counts. The 18s rRNA gene-based nested PCR resulted in 13 (27%) out of 48 wastewater sample positive for Cryptosporidium. C. parvum was the dominated species followed by C. hominis. In ß-giardin based qPCR, all wastewater samples were positive for Giardia Assemblage A and Giardia Assemblage B. The impact of wastewater on the rivers signifies the possible health risks associated with the use of the effluents for agricultural purposes and the receiving water bodies’ use for domestic and recreational activities. Crops irrigated with the river water from the study area gave concentrations of Cryptosporidium and Giardia in spinach samples of 67 to 480 and 3 to 27 per 100 g vegetable respectively. The contamination of the fresh vegetables analysed mainly arose from the use of the effluent from the WWTP B and the receiving river water used for irrigation. In a pilot study researching the effect of chlorine and UV irradiation, distilled water and wastewater samples spiked with environmentally derived Cryptosporidium oocysts and Giardia cysts which were exposed to different doses of chlorine and UV with different exposure times. Viability quantification and detection was done with microscopy and flow cytometry using two vital dyes, namely Syto9+PI and DAPI+PI. Giardia was affected to a much higher degree than Cryptosporidium, both in relation to chlorination and UV, where in the latter case close to zero percent remained viable even after a low dose. Cryptosporidium was found to be resistant to chlorination but responded well to high UV doses. This is in line with similar earlier investigations done in drinking water. In the comparative assessment between the vital dyes, DAPI+PI dyes gave a lower mean percentage viability value than Syto9+PI; and flow cytometry gave a higher mean percentage value than microscopy. The presence of Cryptosporidium and Giardia in all treated effluents from all investigated wastewater treatment plants exceeded World Health Organisation’s (WHO) risk-based values and South African National Standard’s (SANS:241) limit for raw water and irrigation respectively. Enforced regulations and alternative wastewater treatment methods may ensure a higher level of safety for the environment. Additional information on the occurrence of Cryptosporidium and Giardia in wastewater samples, which is currently lacking or limited in South Africa, will further increase our understanding in formulating risk assessment strategies to map out the level of infection to exposed population</jats:p

Efficiency of chlorine and UV in the inactivation of Cryptosporidium and Giardia in wastewater.

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

Mean and SD of viability (%) of (oo)cysts after exposure to chlorine and UV irradiation using flow cytometry.

Mean and SD of viability (%) of (oo)cysts after exposure to chlorine and UV irradiation using flow cytometry.</p

Mean percentages of viable <i>Cryptosporidium</i> and <i>Giardia</i> for different UV doses using microscopy.

Mean percentages of viable Cryptosporidium and Giardia for different UV doses using microscopy.</p

Mean and SD of viability (%) after exposure to chlorine and UV irradiation using microscopy.

Mean and SD of viability (%) after exposure to chlorine and UV irradiation using microscopy.</p

Comparison between the percentages of viability of (oo)cysts when using microscopy and flow cytometer for UV irradiations.

Comparison between the percentages of viability of (oo)cysts when using microscopy and flow cytometer for UV irradiations.</p

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

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).</p