Cryptosporidium is a protozoan parasite that infects humans and various animal species. The environmental stability and the low infectious dose of Cryptosporidium facilitate its transmission by water and food. Discharge of untreated wastewater may result in waterborne or foodborne Cryptosporidium outbreaks, therefore a suitable treatment may prevent its dissemination. Most studies on the prevalence of Cryptosporidium oocysts in wastewater have reported a concentration range between 10 and 200 oocysts/L and a prevalence of 6 to 100%. Activated sludge has been found to be ineffective for the removal of Cryptosporidium oocysts. Stabilization ponds and constructed wetlands are efficient for the reduction of Cryptosporidium from wastewater, especially when the retention time is longer than 20 days at suitable sunlight and temperature. High rate filtration and chlorine disinfection are inefficient for the reduction of Cryptosporidium from effluents, whereas ultrafiltration and UV irradiation were found to be very efficient for the reduction of Cryptosporidium oocysts. Adequate tertiary treatment may result in high quality effluent with low risk of Cryptosporidium for unrestricted irrigation and other non-potable applications.

INTRODUCTION

Cryptosporidium is a protozoan parasite that infects humans and various animal species. The occurrence of outbreaks of cryptosporidiosis have increased awareness concerning the public health significance of Cryptosporidium. The average diameter of Cryptosporidium oocysts is 4–6 μm. The oocyst of Cryptosporidium is protected by an outer wall that allows it to persist in the environment for long periods of time, which facilitates its environmental transmission through contaminated water and food. Human infections are mostly caused either by C. hominis or C. parvum, the cattle genotype. Other species of Cryptosporidium, such as C. meleagridis, C. felis, and C. canis, may occasionally infect humans (Gatei et al. 2002; Plutzer & Karanis 2007; Xiao 2010). These genotypes may infect immunocompromised individuals. The concentration and prevalence of Cryptosporidium oocysts in sewage is influenced by the infection and excretion rates in the population served, and by the contribution of infected domestic animals to the Cryptosporidium load in domestic sewage. However, rainfall may result in the dilution of oocysts in wastewater. Routes of Cryptosporidium transmission include person-to-person, contact with infected animals, recreational water, especially in swimming pools, drinking water and contaminated food (Long et al. 2002; Rose et al. 2002; Pönka et al. 2009; Baldursson & Karanis 2011). Two factors may be responsible for the transmission of Cryptosporidium through the water route: the environmental stability of oocysts and the low infectious dose of the parasite.

Most of the reported waterborne outbreaks of cryptosporidiosis have been in developed countries such as North America, the United Kingdom (UK), Europe, Australia, New Zealand and Japan (Baldursson & Karanis 2011). The waterborne outbreaks were caused by the consumption of contaminated drinking water from surface sources or ground sources and swimming pools (Yoder & Beach 2007; Yoder et al. 2008; Baldursson & Karanis 2011). Cryptosporidium oocysts can enter drinking water sources through sewage contamination, failure of drinking water treatment plants or leakage into the distribution system. As the oocysts of Cryptosporidium are very resistant to chlorine disinfection, in some waterborne outbreaks the water quality complied with the fecal coliform guideline (Craun et al. 1998). The cryptosporidiosis outbreak with the highest number of cases occurred in Milwaukee, Wisconsin, USA in 1993, and involved about 400,000 infected individuals; this outbreak was the result of runoff of wastes due to heavy rain and failure of the drinking water plant to handle heavy contamination (MacKenzie et al. 1994).

The main purpose of advanced wastewater treatment is to reduce the levels of pathogens in order to prevent their environmental transmission. The wastewater treatment applied is governed by the planned application of the effluents and the public health risks involved. Wastewater treatment consists of physical, biological, and chemical processes for the removal of suspended solids, organic materials, and pathogenic microorganisms. The nature of pathogenic microorganisms determines their removal/reduction efficiency by the wastewater treatment process. Advanced wastewater treatment is applied, in particular, in arid regions, where treated effluents are used for irrigation and other non-potable applications. Efficient wastewater treatment is important to prevent the dissemination of pathogenic protozoan parasites by irrigated crops and water bodies that receive the treated wastewater effluents.

PREVALENCE OF CRYPTOSPORIDIUM IN WASTEWATER

Detection of oocysts in stools is taken as a sign of infection. Shepherd et al. (1988) reported that the median time of Cryptosporidium oocysts shedding from onset of symptoms to the last positive stool specimen was 8 days (range from 2 to 35 days). Infections with Cryptosporidium are associated strongly with the age of patients; Cryptosporidium DNA was detected in 21.8% stool samples of children aged <5 years submitted for examination (Ten Hove et al. 2007). Infected persons shed high concentrations of Cryptosporidium oocysts, which can reach up to 105–7 oocysts per gram of feces (Chappell et al. 1999). Similar levels of Cryptosporidium oocysts have been reported for infected calves, where 104–7 oocysts per gram of feces have been detected (Xiao & Herd 1994).

Numerous studies have been conducted around the world to evaluate the prevalence and levels of Cryptosporidium oocysts in raw wastewater. Prior to 1998, most studies applied the sucrose flotation method to purify oocysts of Cryptosporidium from raw wastewater before staining and/or microscopic examination (US Environmental Protection Agency 1996; Bukhari et al. 1998). To improve the recovery efficiency of oocysts from wastewater samples or concentrated water samples, the immune magnetic separation (IMS) method was developed for affinity purification and concentration of oocysts (Bukhari et al. 1998; USEPA method 1622). Detection and enumeration methods have been developed from conventional detection techniques, such as acid-fast Ziehl–Neelsen staining and microscopic examination with a light microscope, or staining with anti-Cryptosporidium oocysts fluorescein isothiocyanate (FITC) labeled monoclonal antibodies (immune fluorescence (IF)) and examination by epi-fluorescent microscope (USEPA method 1623). Microscopic examination of concentrated wastewater or surface water samples may be tedious, time-consuming and require very experienced analysts to accurately identify Cryptosporidium oocysts. In parallel, researchers have attempted to develop alternative methods with greater specificity and sensitivity than the existing methods (Morgan et al. 1998; Guy et al. 2003; Zhou et al. 2003; Feng et al. 2009). Studies have demonstrated that the polymerase chain reaction (PCR) and more recently real time PCR have proved to be more sensitive than the indirect fluorescent antibody (IFA) method for the detection of Cryptosporidium oocysts in raw wastewaters (Guy et al. 2003; Zhou et al. 2003; Feng et al. 2009).

Most reviewed studies have not reported on the detection efficiency of Cryptosporidium oocysts in raw wastewater. The reported detection efficiency of Cryptosporidium oocysts from raw wastewater ranged from 5.5 ± 1.3% to as high as 85% (Gennaccaro et al. 2003; Robertson et al. 2006). It is interesting to note that large variations were reported in the recovery efficiency of oocysts from raw wastewater, which ranged from the lowest detection sensitivity values reported for studies using USEPA method 1623, while less reliable methods such as flotation reported the highest (85%) recovery efficiency (Robertson et al. 2000). McCuin & Clancy (2005) evaluated the recovery efficiency of Cryptosporidium oocysts from samples of 250, 500, and 1,000 mL of raw wastewater by IMS and demonstrated a negative influence of the sample volume on the recovery efficiency. The mean recovery efficiency values from 250 mL, 500 mL, and 1,000 mL of raw wastewater were 33%, 31.8%, and 24.3%, respectively. Since the recoveries achieved from wastewater effluents with sodium chloride or sucrose density flotation were low and very variable, those researchers suggested that the IMS technology is suitable for the efficient recovery of Cryptosporidium oocysts from raw wastewater (McCuin & Clancy 2005).

Table 1 presents the results of 25 studies on the prevalence and concentration of Cryptosporidium oocysts from 13 countries from five continents. The prevalence of Cryptosporidium oocysts in raw wastewater ranged from 0 (where samples contained oocysts below the detection level) to 100% (where Cryptosporidium oocysts were detected in all samples analyzed). Differences in the prevalence of Cryptosporidium oocysts were observed in various studies even in the same country. For example, in Brazil three studies were reviewed, in which Cryptosporidium oocyst prevalence was reported in 6.4, 58.3, and 100% of the samples (Santos et al. 2004; Cantusio Neto et al. 2006; Hachich et al. 2013). In the USA, six studies were reviewed and the reported prevalence ranged from 27.9 to 78% (Mayer & Palmer 1996; Gennaccaro et al. 2003; Zhou et al. 2003; Clancy et al. 2004; Harwood et al. 2005).

Table 1

Prevalence and concentration of Cryptosporidium oocysts in raw wastewater

Country Samples # Cryptosporidium oocysts/L (range) % positive Recovery efficiency Reference 
Brazil 1–560a 100 NRb Santos et al. (2004)  
53 0–60,000a 6.4 46.2 Cantusio Neto et al. (2006)  
24 <2.5–2,700a 58.3 NR Hachich et al. (2013)  
China 90 NR 70 NR Feng et al. (2009)  
22 240 ± 88a 100 NR Fu et al. (2010)  
Germany 206 (0–1,745)a 31.1  Gallas-Lindemann et al. (2013)  
51 50–1,280a 100 NR Ajonina et al. (2012)  
Italy 4.5 ± 0.8a 100 NR Carraro et al. (2000)  
Japan 73 8–50a 10 14 Suwa & Suzuki (2001)  
Kenya 11 12.5–72.97a 18.2 NR Grimason et al. (1993)  
Malaysia 24 1–80a 50 NR Lim et al. (2007)  
Norway 32 100–1,100 (200)a 80 NR Robertson et al. (2006)  
Spain 10 45.7 ± 7.5a NR 44.1 ± 10.8 Reinoso et al. (2008)  
46 103–139a 100 NR Montemayor et al. (2005)  
48 8–970a 100 NR Castro-Hermida et al. (2008)  
S. Africa 56 0–150 (31.7)a 60.7 NR Dungeni & Momba (2010)  
Sweden 19 5 (3–220)a 26.3 15 ± 4.6 Ottoson et al. (2006)  
UK 33 111 ± 127a NR 85 ± 18 Robertson et al. (2000)  
USA 179 NRc 27.9 NR Zhou et al. (2003)  
95 <2–24a 29.5 NR Clancy et al. (2004)  
18 <5.5–263a 78 5.5 ± 1.3 Gennaccaro et al. (2003)  
30 3–21a 74 NR Harwood et al. (2005)  
Country Samples # Cryptosporidium oocysts/L (range) % positive Recovery efficiency Reference 
Brazil 1–560a 100 NRb Santos et al. (2004)  
53 0–60,000a 6.4 46.2 Cantusio Neto et al. (2006)  
24 <2.5–2,700a 58.3 NR Hachich et al. (2013)  
China 90 NR 70 NR Feng et al. (2009)  
22 240 ± 88a 100 NR Fu et al. (2010)  
Germany 206 (0–1,745)a 31.1  Gallas-Lindemann et al. (2013)  
51 50–1,280a 100 NR Ajonina et al. (2012)  
Italy 4.5 ± 0.8a 100 NR Carraro et al. (2000)  
Japan 73 8–50a 10 14 Suwa & Suzuki (2001)  
Kenya 11 12.5–72.97a 18.2 NR Grimason et al. (1993)  
Malaysia 24 1–80a 50 NR Lim et al. (2007)  
Norway 32 100–1,100 (200)a 80 NR Robertson et al. (2006)  
Spain 10 45.7 ± 7.5a NR 44.1 ± 10.8 Reinoso et al. (2008)  
46 103–139a 100 NR Montemayor et al. (2005)  
48 8–970a 100 NR Castro-Hermida et al. (2008)  
S. Africa 56 0–150 (31.7)a 60.7 NR Dungeni & Momba (2010)  
Sweden 19 5 (3–220)a 26.3 15 ± 4.6 Ottoson et al. (2006)  
UK 33 111 ± 127a NR 85 ± 18 Robertson et al. (2000)  
USA 179 NRc 27.9 NR Zhou et al. (2003)  
95 <2–24a 29.5 NR Clancy et al. (2004)  
18 <5.5–263a 78 5.5 ± 1.3 Gennaccaro et al. (2003)  
30 3–21a 74 NR Harwood et al. (2005)  

adetection method immune fluorescence (IF).

bnot reported.

cPCR.

The concentration of Cryptosporidium oocysts detected in raw wastewater ranged from 0 (below the detection limit) to 60,000 oocysts/L. The highest values for Cryptosporidium in raw wastewater were reported from Brazil (Cantusio Neto et al. 2006). The dominant values reported for the concentration of Cryptosporidium in raw wastewater were from 10 to 200 oocysts/L (Table 1). Dungeni & Momba (2010) studied the concentration and prevalence of Cryptosporidium oocysts in four wastewater treatment plants in South Africa. A concentration of up to 150 oocysts/L was detected in the influent of the wastewater treatment plant with up to 79% prevalence level.

Several factors may influence the prevalence and concentration of Cryptosporidium in wastewater. The incidence of cryptosporidiosis in the community may influence the levels of Cryptosporidium oocysts in wastewater. In the USA, the reported cases of cryptosporidiosis for the years 2009 and 2010 were from a high level of 17.4 in 100,000 in Wisconsin in 2010 to a low level of 0.6 in 100,000 in 2009 in Mississippi. The high incidence was reported in states that experience cryptosporidiosis outbreaks, or have more efficient reporting systems (Yoder et al. 2012). In Europe, cryptosporidiosis is notifiable through the European Basic Surveillance Network. In 2005, 7,960 cryptosporidiosis cases were reported from 16 countries, which makes a crude incidence rate of 1.9 cases per 100,000. A clear peak of cryptosporidiosis was recorded in the autumn of 2005. The highest incidence was reported in Ireland with a rate of 13.7 followed by the UK with an incidence rate of 9.3 cases per 100,000. No cases of cryptosporidiosis were reported for eight out of the 16 European countries included in the study (Semenza & Nichols 2007). The incidence rate of Cryptosporidium may be influenced by the socio-economic and hygienic status of the community. Garvey & McKeown (2009) reported higher incidence rates of Cryptosporidium in rural than in urban settings.

The seasonal distribution of Cryptosporidium in wastewater has been examined in various parts of the world (Table 2). Ajonina et al. (2012) showed that oocysts of Cryptosporidium are predominant during autumn and winter in Germany. In Ireland, Cheng et al. (2012) reported high abundance of Cryptosporidium in sewage, especially in tourist towns; sudden season-dependent traveler influx may be a potential source of pathogens. Cryptosporidium was detected in over 93% of wastewater samples in summer, autumn, and winter; however, the detection frequency in spring was 69%. In Spain, the highest numbers of oocysts were detected in spring and summer in influent and effluent wastewater samples (Castro-Hermida et al. 2008), while Montemayor et al. (2005) demonstrated the occurrence of two peaks in oocyst concentration, one in early spring and another in autumn. Robertson et al. (2006) reported no pattern of seasonality in the occurrence of Cryptosporidium in Norway. A study conducted in Italy has shown that the oocysts of Cryptosporidium were detected during the spring in raw wastewater (Caccio et al. 2003).

Table 2

Seasonality of Cryptosporidium prevalence in wastewater

Country Season Reference 
Germany Autumn and winter Ajonina et al. (2012)  
Ireland Summer, autumn, and winter Cheng et al. (2012)  
Spain Spring and summer Castro-Hermida et al. (2008)  
Spring and autumn Montemayor et al. (2005)  
Italy Spring Caccio et al. (2003)  
Country Season Reference 
Germany Autumn and winter Ajonina et al. (2012)  
Ireland Summer, autumn, and winter Cheng et al. (2012)  
Spain Spring and summer Castro-Hermida et al. (2008)  
Spring and autumn Montemayor et al. (2005)  
Italy Spring Caccio et al. (2003)  

REMOVAL OF CRYPTOSPORIDIUM BY SECONDARY WASTEWATER TREATMENT

Various groups of pathogenic microorganisms (viruses, bacteria, protozoan parasites, and worms) may be present in wastewater effluents. Therefore, to prevent the environmental transmission of pathogens, treatment is applied to reduce or inactivate these pathogens in effluent before discharge. The wastewater treatment applied is governed by the planned application of the effluents and the public health risks involved. Wastewater treatment consists of physical (sedimentation and filtration), biological (activated sludge, trickling filters, and stabilization ponds), and chemical processes (chlorine, ozone, and chlorine dioxide disinfection). Multi-barrier treatment (mostly consisting of sedimentation, biological treatment, filtration, and disinfection) is applied to wastewater when reclaimed wastewater effluents are utilized for unrestricted irrigation of food crops or for groundwater recharge.

A prevalence of 12% was demonstrated for Cryptosporidium oocysts in activated sludge-treated effluent at a concentration range of 0.05 to 1.6 oocysts/L in Japan (Suwa & Suzuki 2001) (Table 3). A study on the prevalence and removal efficiency of Cryptosporidium oocysts by activated sludge in four wastewater treatment plants in South Africa has shown a concentration of up to 40 oocysts/L in effluent samples and a prevalence of up to 86% of oocysts in effluent samples. The researchers reported no significant differences in the number of oocysts isolated from the effluent samples (P ≥0.05) of larger and smaller plants (Dungeni & Momba 2010).

Table 3

Prevalence of Cryptosporidium in secondary activated sludge-treated effluents

Country Wastewater treatment Prevalence (%) Concentration oocysts/L Reference 
Italy Activated sludge NRa 0.21 ± 0.06 Carraro et al. (2000)  
Japan Activated sludge 12 0.05–1.6 Suwa & Suzuki (2001)  
S. Africa Activated sludge 50–79 1–150 Dungeni & Momba (2010)  
Spain Activated sludge NR 2–390 Castro-Hermida et al. (2008)  
Malaysia Extended aeration 25% 20–40 Lim et al. (2007)  
USA Activated sludge 58.5 <0.1–40.8 Clancy et al. (2004)  
Activated sludge 84%  Harwood et al. (2005)  
Activated sludge 83 <12.8–345 Gennaccaro et al. (2003)  
Spain Activated sludge 100 1.8–5.6 Montemayor et al. (2005)  
Ireland Activated sludge 97% 10–32 Graczyk et al. (2009)  
Oxidation ditch NR 4 ± 2 Cheng et al. (2009)  
Activated sludge NR 8 ± 3.6 Cheng et al. (2009)  
England Activated sludge 15.4–46.6 10–69 Bukhari et al. (1997)  
Sweden Activated sludge 5.2  Ottoson et al. (2006)  
Country Wastewater treatment Prevalence (%) Concentration oocysts/L Reference 
Italy Activated sludge NRa 0.21 ± 0.06 Carraro et al. (2000)  
Japan Activated sludge 12 0.05–1.6 Suwa & Suzuki (2001)  
S. Africa Activated sludge 50–79 1–150 Dungeni & Momba (2010)  
Spain Activated sludge NR 2–390 Castro-Hermida et al. (2008)  
Malaysia Extended aeration 25% 20–40 Lim et al. (2007)  
USA Activated sludge 58.5 <0.1–40.8 Clancy et al. (2004)  
Activated sludge 84%  Harwood et al. (2005)  
Activated sludge 83 <12.8–345 Gennaccaro et al. (2003)  
Spain Activated sludge 100 1.8–5.6 Montemayor et al. (2005)  
Ireland Activated sludge 97% 10–32 Graczyk et al. (2009)  
Oxidation ditch NR 4 ± 2 Cheng et al. (2009)  
Activated sludge NR 8 ± 3.6 Cheng et al. (2009)  
England Activated sludge 15.4–46.6 10–69 Bukhari et al. (1997)  
Sweden Activated sludge 5.2  Ottoson et al. (2006)  

aNot reported.

In Spain, a concentration range of 2 to 390 oocysts/L of Cryptosporidium oocysts was reported in activated sludge effluent (Table 3). Even though the concentration of oocysts in the final effluent throughout the year did not differ significantly among the wastewater treatment plants (WWTP), there is a possibility that oocysts were stably removed in WWTP. It was also found that the concentration of oocysts per liter was higher in the treated than in the untreated wastewater (Castro-Hermida et al. 2008). On the other hand, in the USA, 55 out of 94 (58.5%) activated sludge-treated effluent samples were found positive for Cryptosporidium at a concentration range of <0.1 to 40.8 oocysts/L. It was reported that in many cases the concentration of Cryptosporidium oocysts in effluents was found to be higher than that detected in the influent samples. Four reasons were mentioned by the authors as possibly contributing to this observation: (1) the low levels of Cryptosporidium detected in the influent make it difficult to show significant log10 differences; (2) the improvement of recovery of oocysts in cleaner water matrices than that obtained in raw wastewater; (3) the low removal efficiency of Cryptosporidium in secondary and tertiary treatments; and (4) aggregated oocysts with organic material may be dispersed through the treatment processes, resulting in greater numbers in the effluent. Robertson et al. (2006) reported a removal efficiency of 0 to 50% for Cryptosporidium by activated sludge treatment and a concentration range of 230 to 3,300 oocysts/L. The authors also reported that in some of the effluent samples, the numbers of parasites detected was greater than those detected in the influent samples (Robertson et al. 2006). They explained the results as an artifact of higher method recovery efficiencies with the cleaner effluent samples or a reflection of temporal fluctuations in parasite concentrations, the lack of pairing of the samples, the uneven distribution of parasites in the sample matrices, or a combination of these factors.

In a study conducted by Lim et al. (2007) in Malaysia, the researchers reported that a secondary treatment process in which extended aeration was employed resulted in 73% reduction of the oocysts; however, a treatment process that encompassed an aerated lagoon did not reduce the concentration of Cryptosporidium oocysts significantly. Treated wastewater effluents contained Cryptosporidium oocysts a a concentration of 20 to 40 oocysts/L in three out of 12 (25%) effluent samples examined (Lim et al. 2007). The results are in agreement with previously reported results on the inefficient removal of Cryptosporidium by secondary wastewater treatment. Harwood et al. (2005) reported that following biological treatment, the frequency of detection of Cryptosporidium increased from 75% in the influent samples to 84%. The increase was explained to be due to the more sensitive detection in secondary effluent (21 to 94 oocysts/100 L) (Harwood et al. 2005). Gennaccaro et al. (2003) studied the removal efficiency of Cryptosporidium oocysts from wastewater by activated sludge treatment. The removal efficiency was estimated by IFA and cell-culture infectivity. A prevalence of 83% was recorded in secondary effluent at a concentration range of <12.8 to 345 oocysts/100 L, and the arithmetic mean was 112 ± 153 oocysts/100 L. The arithmetic mean of infectious oocysts in secondary effluent was 37 ± 28 oocysts/100 L. The results indicate that activated sludge treatment is not efficient for the removal of Cryptosporidium from wastewater (Gennaccaro et al. 2003).

Costán-Longares et al. (2008) studied the comparative removal of indicator and pathogenic microorganisms by wastewater treatment processes. Their findings have shown that the reductions in the numbers of viable Cryptosporidium oocysts were significantly lower than those of all other parameters and only approached by the reduction of sulphite reducing clostridia. The prevalence of Cryptosporidium in secondary effluent was 94% and the mean values of viable Cryptosporidium oocysts were 1.9 log10 oocysts/100 L (Costán-Longares et al. 2008).

The removal efficiency of Cryptosporidium by sewage treatment processes depends upon their concentration in the influent and the processes applied (Cheng et al. 2009). The removal by typical sewage treatments is critical, and never guarantees a complete abatement of these protozoa. Primary and secondary sedimentation allows a variable removal due to the difference between the sedimentation velocities of oocysts with respect to the process (Enriquez et al. 1995; Withmore & Robertson 1995). Medema et al. (1998) pointed out that the ability of sedimentation to settle cysts and oocysts is related to their attachment to particles. The removal efficiency of the activated sludge process for Cryptosporidium is related to the clarification process: the flocculated material in the activated sludge was believed to contribute to the removal, acting as a ‘settling blanket’ (Enriquez et al. 1995; Withmore & Robertson 1995).

CRYPTOSPORIDIUM REMOVAL FROM WASTEWATER BY NATURAL TREATMENT METHODS

Biological treatment of domestic wastewater by natural systems can be accomplished by either constructed wetland (CWL) or waste stabilization ponds (WSPs). CWL and WSP systems are applied to treat wastewater of small communities and in regions were land is not limited. Two types of CWLs, subsurface flow (SSF) and free-surface flow (FSF), were evaluated for their efficiency in removing Cryptosporidium from domestic wastewater. The mechanisms responsible for the reduction of Cryptosporidium may be influenced by the configuration of the CWL, biological factors such as predation, microbial activity and chemical factors such as oxidation reactions, adsorption and exposure to toxins excreted from plants and bacteria. Additional mechanisms for pathogen reduction in CWLs include sedimentation, natural die-off, inactivation or death related to temperature, inactivation or death related to unfavorable water chemistry, biofilm interaction, mechanical filtration, exposure to biocides and UV radiation. Pathogen removal in CWL has also been shown to correlate with hydraulic retention time (Kadlec & Knight 1996). Table 4 presents results of Cryptosporidium oocysts reduction by extensive treatment methods.

Table 4

Removal of Cryptosporidium from wastewater by natural treatment processes

Country Wastewater treatment Removal %/log10 Reference 
Ireland Wetlands (SSF) 97.5% Graczyk et al. (2009)  
Kenya Stabilization pond 100% Grimason et al. (1993)  
Spain Oxidation pond 97% Araki et al. (2001)  
Waste stabilization pond 40% Reinoso & Bécares (2008)  
Anaerobic pond 4.6 Reinoso et al. (2011)  
Facultative pond 5.2 
maturation 3.7 
Facultative pond 2.81 Reinoso et al. (2008)  
Surface flow wetland (SFW) 3.21 
Subsurface flow wetland (SSFW) 2.13 
USA Duckweed pond 89% Falabi et al. (2002)  
Subsurface flow wetland (SSFW) 64.2% Thurston et al. (2001)  
Country Wastewater treatment Removal %/log10 Reference 
Ireland Wetlands (SSF) 97.5% Graczyk et al. (2009)  
Kenya Stabilization pond 100% Grimason et al. (1993)  
Spain Oxidation pond 97% Araki et al. (2001)  
Waste stabilization pond 40% Reinoso & Bécares (2008)  
Anaerobic pond 4.6 Reinoso et al. (2011)  
Facultative pond 5.2 
maturation 3.7 
Facultative pond 2.81 Reinoso et al. (2008)  
Surface flow wetland (SFW) 3.21 
Subsurface flow wetland (SSFW) 2.13 
USA Duckweed pond 89% Falabi et al. (2002)  
Subsurface flow wetland (SSFW) 64.2% Thurston et al. (2001)  

A study was conducted to compare the removal efficiency of Cryptosporidium by SSF and FSF wetlands. The concentration of Cryptosporidium in the influent samples varied from 10 to 118 oocysts/L. A removal of 97.5% was recorded for Cryptosporidium in SSF wetlands, whereas no reduction was observed in FSF wetlands where greater concentration of Cryptosporidium was observed in the effluent as compared to the influent (Table 4). The concentration of Cryptosporidium was 10 to 32 oocysts/L in the influent samples compared to 22 to 78 oocysts/L in the effluent samples (Graczyk et al. 2009).

Reinoso et al. (2008) studied the comparative removal of protozoan parasites and indicator microorganisms in facultative pond (FP), surface flow (SF) wetland, and SSF wetland, in Spain. The concentration of Cryptosporidium oocysts in the effluent samples was 45.7 oocysts/L and a prevalence of 7.55% was recorded. Removal efficiency values of 2.81, 3.21, and 2.13 log10 were recorded for Cryptosporidium oocysts in FB, SF, and SSF, respectively. No significant difference was observed between the removal of Cryptosporidium during the summer and winter months (Reinoso et al. 2008). It is worth noting that the removal rates were not determined by infectivity of Cryptosporidium.Thurston et al. (2001) studied the removal efficiency of oocysts by SSF-constructed wetland from secondary effluent. The concentration of the Cryptosporidium oocysts ranged from 0.9 to 108.9/100 L in the influent and <1.0 to 49.9/100 L in the effluent. The reduction ranged from 31.7 to 98.3% and the average removal was 64.2% (Thurston et al. 2001). Sedimentation is thought to be one of the major mechanisms of microbial reduction from wetlands used for wastewater treatment. This observation was supported by Karim et al. (2004), who reported concentrations of one to three orders of magnitude greater in the sediment compared to the water column for Giardia cysts and Cryptosporidium oocysts (Karim et al. 2004).

Cryptosporidium oocysts decreased by 89% using duckweed ponds, with an average number of 1.58 oocysts/L in the influent and 1.17 oocysts/L in the effluent. The duckweed pond was more effective in reducing the number of protozoan parasites (Giardia 98%; Cryptosporidium 89%) than indicator bacteria (total coliform 61%, fecal coliform 62%) or coliphages (Falabi et al. 2002). Therefore, the researchers concluded that the removal of protozoan parasites in the pond appeared to be related to their size.

WSPs are large, man-made water bodies in which wastewater is treated by natural processes. There are three types of ponds: (1) anaerobic, (2) facultative, and (3) aerobic (maturation), each with different treatment and design characteristics. Anaerobic ponds are built to a depth of 2 to 5 m and have a relatively short retention time of 1 to 7 days. Facultative ponds are constructed to a depth of 1 to 2.5 m and have a retention time between 5 and 30 days. Aerobic ponds are usually between 0.5 and 1.5 m deep with a retention time of 15 to 20 days. Ideally, several aerobic ponds can be built in series to provide a high level of pathogen removal. Reduction of pathogenic parasites in WSPs results from numerous processes, such as sedimentation, sunlight, predation, natural die-off, hydraulic residence time, and environmental variables, such as temperature, pH, dissolved oxygen, water potential, and ammonia.

Table 4 also presents results on the reduction of Cryptosporidium oocysts by WSPs. Reinoso et al. (2011) studied the mechanisms for the removal of parasites from waste stabilization ponds in northern Spain. They compared the removal efficiency of parasites and indicator microorganisms by anaerobic, facultative, and maturation ponds. Cryptosporidium oocysts were reduced by an average of 96%. The anaerobic ponds showed significantly higher surface removal rates (4.6, 5.2, and 3.7 log oocysts removed m−2 day−1, respectively) than facultative and maturation ponds. Sunlight and water physicochemical conditions were the main factors influencing the removal of C. parvum oocysts both in the anaerobic and maturation ponds, whereas other factors like predation or natural mortality were more important in the facultative ponds. The results showed that sedimentation, the most commonly proposed mechanism for oocyst removal, was found to be negligible, ranging from zero in the maturation pond to 5.56% in the facultative pond. Reinoso & Bécares (2008) studied factors affecting the reduction of C. parvum oocysts in a waste stabilization pond. They evaluated the effects of sunlight, pond depth, and type of treatment pond on oocyst viability by an assay based on the exclusion or inclusion of two vital dyes, 4′,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI). Sunlight exposure was the most influential factor for oocyst inactivation. A 40% reduction was observed after 4 days of exposure to sunlight conditions compared with dark conditions. Inactivation rates suggested that the facultative ponds were the most aggressive environment for oocyst inactivation, followed by the maturation ponds and the anaerobic ponds (Reinoso & Bécares 2008).

Araki et al. (2001) studied the effect of high-rate algal ponds on the infectivity of C. parvum. A reduction of 97% in the infectivity of Cryptosporidium oocysts in neonatal mice was recorded within 6 days of exposure. The researchers suggested that pH, ammonia, and/or sunlight were major factors for the inactivation of oocysts in wastewater.

Gómez-Couso et al. (2009) studied the effect of water turbidity on the inactivation efficacy of Cryptosporidium by the solar water disinfection (SODIS) method. Polyethylene terephthalate (PET) bottles containing the contaminated turbid waters were exposed to full sunlight for 4, 8, and 12 h. SODIS significantly reduced the potential viability of C. parvum oocysts to increase the percentage of oocysts that took up the dye PI. The opposite correlation was demonstrated between the water turbidity level and the loss of viability of the oocysts (Gómez-Couso et al. 2010). These results are in agreement with data reported by Nasser et al. (2007), who demonstrated that sunlight enhances the reduction of infectivity of C. parvum and at the end of the 7-day study period a 3 log reduction in infectivity was measured in the sunlight- and seawater-exposed oocysts.

A study conducted on 11 WTPs in Kenya demonstrated that oocyst levels in the influent wastewater samples ranged from 12.5 to 72.97 oocysts/L, whereas no Cryptosporidium oocysts were detected in any of the final effluents of the studied pond systems (Grimason et al. 1993).

REDUCTION OF CRYPTOSPORIDIUM BY TERTIARY TREATMENT

Tertiary or advanced wastewater treatment is employed for the removal of contaminants that are not efficiently removed by secondary treatment and are harmful to the environment and public health. In many countries, wastewater is treated for reuse purposes to increase the water budget either for unrestricted crop irrigation, stream restoration, or other non-potable applications. To produce high quality effluent in intensive WTPs, a common treatment train consists of chemical coagulation of the secondary clarified effluent, followed by sedimentation, rapid sand filtration, and final disinfection (Rose 2007). Membrane ultrafiltration has also been used for tertiary treatment of secondary effluents and has proved to be efficient in the removal of Cryptosporidium oocysts (Lonigro et al. 2006). Disinfection normally involves the injection of a chlorine solution at the head end of a chlorine contact basin. Ozone and UV irradiation can also be used for disinfection. This level of treatment is assumed to produce an effluent free from detectable pathogens.

The prevalence of Cryptosporidium oocysts in tertiary effluents was reported by Harwood et al. (2005). The studied wastewater treatment train consisted of activated sludge, filtration and, in some cases, flocculation and chlorination. The concentration of Cryptosporidium oocysts did not decrease substantially in comparison to their prevalence in untreated influent and was recorded at 70% with a concentration range of 0.01 to 1 oocysts/100 L (Harwood et al. 2005) (Table 5). One explanation for the poor removal was an artifact related to the greater sensitivity of the detection method as the quality of the effluents is improved through treatment stages. The researchers reported that infectious oocysts were present in 20% of the reclaimed effluent samples (Harwood et al. 2005). Flocculation and sand filtration produced tertiary effluent with a concentration of Cryptosporidium consistently below 0.4 oocysts/L and a removal efficiency of 1.69 log (Fu et al. 2010). The results from a study conducted in Spain have shown that all samples of tertiary treated effluent (sand filtration, UV irradiation, and chlorination) were found positive for oocysts of Cryptosporidium with a mean value of 0.4 oocysts/L (Montemayor et al. 2005). Removal efficiencies of viable oocysts by sand filtration, UV, and chlorination were found to be 97.9% (Montemayor et al. 2005) (Table 5).

Table 5

Prevalence of Cryptosporidium in tertiary-treated effluents

Country Concentration oocysts/L Prevalence (%) Wastewater treatment Reference 
USA 0.01–1 70%a Filtration, flocculation, and chlorination Harwood et al. (2005)  
<1.8–178 67 Filtration and disinfection Gennaccaro et al. (2003)  
<0.008–0.226 18.8 Filtration and disinfection Clancy et al. (2004)  
Spain 0.14–0.22 75 Sand filtration + UV Montemayor et al. (2005)  
Ireland 4 ± 2  Biofilm-coated filter Cheng et al. (2009)  
<0.4 41 Flocculation and sand filtration 
<0.2b Membrane filtration 
Italy Ultrafiltration Lonigro et al. (2006)  
China 0–0.4 41 Flocculation and sand filtration Fu et al. (2010)  
Ultrafiltration 
Country Concentration oocysts/L Prevalence (%) Wastewater treatment Reference 
USA 0.01–1 70%a Filtration, flocculation, and chlorination Harwood et al. (2005)  
<1.8–178 67 Filtration and disinfection Gennaccaro et al. (2003)  
<0.008–0.226 18.8 Filtration and disinfection Clancy et al. (2004)  
Spain 0.14–0.22 75 Sand filtration + UV Montemayor et al. (2005)  
Ireland 4 ± 2  Biofilm-coated filter Cheng et al. (2009)  
<0.4 41 Flocculation and sand filtration 
<0.2b Membrane filtration 
Italy Ultrafiltration Lonigro et al. (2006)  
China 0–0.4 41 Flocculation and sand filtration Fu et al. (2010)  
Ultrafiltration 

a20% of the detected oocysts were infectious.

bBelow detection limit.

Membrane technologies have significant potential to produce high quality effluents and ultrafiltration can achieve high removal of protozoan cysts by physical sieving. Lonigro et al. (2006) evaluated the removal efficiency of Cryptosporidium oocysts from secondary effluents by membrane ultrafiltation and demonstrated high removal of parasites from the effluents, with all collected permeate samples testing negative for Cryptosporidium oocysts. These results were supported by Fu et al. (2010), who demonstrated that the levels of Cryptosporidium oocysts in all the ultrafiltration-treated effluents were below the detection limit and up to 1.84 log10 oocyst reduction was demonstrated. Similar results were recorded for the removal efficiency of Cryptosporidium oocysts by microfiltration and ultrafiltration, which resulted in a reduction of >7 log10 (Hirata & Hashimoto 1998).

The results presented suggest that membrane ultrafiltration provides high log removal of Cryptosporidium oocysts, indicating its usefulness for removal of pathogenic protozoan Cryptosporidium oocysts from wastewater.

Disinfection is considered the last barrier to prevent the environmental transmission of Cryptosporidium oocysts by wastewater effluents. The concentration X time (CT) product of free chlorine per 1 log reduction in infectivity of Cryptosporidium was estimated to be in the range of 800 to 900 mg min/L (Hirata et al. 2001); while a CT product of 2,700 mg min/L was needed for 3 log reduction of animal infectivity of Cryptosporidium oocysts (Hirata et al. 2001). The resistance of Cryptosporidium to free chlorine and monochloramine was also reported by Rennecker et al. (2000), who reported that a CT of about 2,000 mg min/L and 12,000 mg min/L were required to reach inactivation of 99.9% of C. parvum by chlorine and monochloramine, respectively. Similar results on the resistance of Cryptosporidium to free chlorine were reported earlier by Korich et al. (1990). Alternative disinfection methods were evaluated in order to replace chlorine as the sole disinfectant and to efficiently inactivate oocysts present in tertiary effluent. Ozone was proved to be efficient for the inactivation of Cryptosporidium, where greater than 90% inactivation, as measured by infectivity, was achieved by treating Cryptosporidium oocysts with 1 ppm of ozone (1 mg/L) for 5 min (Rennecker et al. 2000). The CT products of ozone per 1 log reduction in infectivity were 3 mg min/L. However, a 99% inactivation of C. parvum oocysts was achieved with an ozone CT of 5 mg × min/L at 20 °C. When used as primary disinfectant, ozone resulted in a strong synergy and the inactivation rates of C. parvum by free chlorine and monochloramine were 1.1–2.8 and 2.4–9.2 times faster, respectively, than the corresponding primary inactivation rate (Rennecker et al. 2000).

In the late 1990s, UV irradiation was found efficient for inactivating Cryptosporidium oocysts (Carpenter et al. 1999). After that, numerous studies demonstrated the effectiveness of low doses of UV irradiation to inactivate Cryptosporidium oocysts in various water matrixes. For example, Morita et al. (2002) showed that a UV dose of 1.0 mWs/cm2 at 20 °C was needed to produce a 2-log10 reduction in infectivity (99% inactivation). No recovery in infectivity following treatment was observed after exposure to fluorescent-light irradiation or storage in darkness. The results indicate that the effects of UV irradiation on C. parvum oocysts as determined by animal infectivity can conclusively be considered irreversible (Morita et al. 2002). These results are in agreement with numerous studies conducted on the inactivation of Cryptosporidium oocysts in water and wastewater. Using either animal infectivity or cell culture, Cryptosporidium was reported as sensitive to low UV radiation doses (Clancy et al. 2004).

CONCLUSIONS

This review paper presents results of studies concerning the prevalence of Cryptosporidium in wastewater and its removal by wastewater treatment processes. Cryptosporidium oocysts have been detected in wastewater at an average concentration of 10 to 200 oocysts/L. Activated sludge is inefficient for the removal of Cryptosporidium oocysts from wastewater and similar results were observed for high-rate sand filtration. UV radiation is the most effective disinfection process for the inactivation of Cryptosporidium. WSPs with a retention time longer than 20 days and SSF wetlands resulted in high removal of Cryptosporidium oocysts from wastewater. Application of efficient wastewater treatment processes may prevent the environmental transmission of Cryptosporidium.

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