Five municipal and domestic wastewater treatment plants, most of which had secondary treatment systems formed by activated sludge, were studied during 2013–2014 in Tehran. The study was done in order to evaluate their efficiency in terms of removal of Cryptosporidium and Giardia by (oo)cyst recovery in effluent samples using immunofluorescence with monoclonal antibodies. Results showed that mean concentrations of cysts in the influent samples always outnumbered mean concentrations of oocysts (883.3 ± 4,16.7–3,191.7 ± 1,067.2 versus 4.8 ± 6.2–83.8 ± 77.3 (oo)cysts/L), and that lower concentrations of (oo)cysts were recorded in summer, and higher levels in autumn, and that the difference was statistically significant (t-test, P <0.05) only in wastewater from slaughterhouses. Results for removal percentages of all the plants ranged from 76.7 to 92.1% for cysts and from 48.9 to 90.8% for oocysts. There was more reduction of (oo)cysts at the urban treatment plant by activated sludge-A2O-sand filtration than at plants with conventional activated sludge and activated sludge-trickling filter, however, this difference was not statistically significant for cysts and oocysts (ANOVA, P > 0.05). Infections in mice inoculated with cysts obtained from urban wastewater effluent demonstrated presence of infectious Giardia cysts. Results demonstrate limited efficiency of conventional wastewater treatment processes at physico-chemical removal of (oo)cysts.

INTRODUCTION

Freshwater shortage is expected to become an ever-increasing problem in the future for many countries. Water deficit may be caused by climate change, such as altered atmospheric patterns, reduced annual precipitation, and overuse of water, a problem that is exacerbated by rises in population (Moe & Rheingans 2006). Every day in rural communities and poor urban centers throughout the world, hundreds of millions of people are suffering because of limited access to clean, safe water. Over 80% of the water used worldwide is not collected or treated, and up to 90% of wastewater in developing countries flows untreated into streams, rivers, and lakes thus threatening human health, food security, and access to safe drinking water (Corcoran et al. 2010). This situation can be overcome if wastewater is treated for reuse as irrigation water for parks and gardens, agriculture and horticulture, as well as other purposes in which water with low level chemical or microbiological contaminant can be applied without contravening water quality standards or threating human health (Kahana & Tal 2000).

The parasites Giardia and Cryptosporidium are major causes of human gastroenteritis. Such parasites are the most common human protozoan pathogens transmitted by water, and as such they present a significant challenge to public health worldwide (Fletcher et al. 2012). They occur in domestic and feral animals as well as in humans (Hunter & Thompson 2005). Infected hosts shed a large number of cysts and oocysts into the environment (Caccio et al. 2003). Both cysts and oocysts are environmentally robust and can survive in aquatic environments for a long period of time and are strongly resistant to physico-chemical stressors (Dumètre et al. 2012). These organisms are highly infective and ingestion by humans of as few as 10 cysts or 30 oocysts can result in an infection (Caccio et al. 2003).

At least 325 human waterborne outbreaks of protozoasis have been reported throughout the world and 50.8% of outbreaks are attributed to Cryptosporidium species (C. parvum and C. hominis) and 40.6% to Giardia duodenalis (Karanis et al. 2007). Recreational waterborne outbreaks caused by C. parvum have also been reported (Karanis et al. 2006). Data from genetic evaluation concludes that waterborne outbreaks were caused by anthropozoonotic Giardia and Cryptosporidium from animal and human wastewater sources (Rose et al. 2002).

Currently, detection of waterborne cysts and oocysts in aquatic environments involves a range of techniques that employ filtering large volumes of sample followed by centrifugation and clarification (either by flotation or immunomagnetic separation) to concentrate (oo)cysts. A variety of other methods have also been used to isolate and enumerate (oo)cysts such as immunofluorescence and polymerase chain reaction (PCR) (Smith & Grimason 2003).

In Iran, there are only a few publications reporting on data related to the presence of Giardia and Cryptosporidium in wastewater (Sharafi et al. 2012). However, many studies have reported data on the molecular prevalence of these parasites in human and domestic animal feces and surface waters (Meamar et al. 2007; Fallah et al. 2008a, 2008b).

The main purpose of this study was to determine the (oo)cyst removal efficiency for three urban wastewater treatment plants (WWTPs) and two slaughterhouse WWTPs (SWWTPs) (all with activated sludge secondary treatment process) by (oo)cyst recovery in both influent and effluent wastewater samples by immuno-parasitological assay, and to access the cysticidal effectiveness of the treatment processes in terms of inactivating Giardia cysts in effluent using eosin exclusion and animal infectivity assays.

MATERIALS AND METHODS

WWTPs

Three human WWTPs and two SWWTPs located in metropolitan and suburban areas of Tehran were chosen for sample collection (Figure 1).

Figure 1

Map of Tehran, Iran, indicating the location of the five WWTPs. WWTPS, municipal wastewater treatment plants; SWWTPs, slaughterhouse wastewater treatment plants.

Figure 1

Map of Tehran, Iran, indicating the location of the five WWTPs. WWTPS, municipal wastewater treatment plants; SWWTPs, slaughterhouse wastewater treatment plants.

The treatment process applied in all five of the plants is described in specific steps and shown in Table 1. One of the plants was located in the west of the capital (Shahrak-e Ekbātān: WWTP1) and in this plant, primary treatment did not include sedimentation, and secondary treatment consisted of activated sludge and A2/O followed by sand filtration. Another of the plants was located in the north-western part of Tehran (Shahrak-e Gharb: WWTP2), and the secondary treatment system in this plant consisted of a conventional activated sludge system. The third municipal plant (Tehran southern wastewater treatment plant: WWTP3) under consideration was located in the south of the city in Shahr-e Ray, an area that will be out of the development limit of Tehran city in the next 25 years. In this plant, secondary treatment consisted of activated sludge and trickling filtration, and disinfection, consisting of chlorination and UV radiation. The overall quality of water produced from each water reclamation facility generally met the Tehran sewerage company's minimum standards for public access reuse and agricultural purposes. These standards are as follows: total N, less than 30 mg per liter; total suspended solids (single-sample maximum), 25 mg per liter or less; moderate turbidity, <10 nephelometric units; fecal coliforms, less than 1,000 per 100 mL; and nematode eggs, <1 per liter. Two SWWTPs were included in the study; these were located in the Tehran suburbs of Robat-Karim (Meisam-robat-dam = SWWTP4) and Shahriyar (Dam-pak = SWWTP5). The primary treatment carried out at SWWTPs was sedimentation, and the secondary treatment consisted of activated sludge and oxidation with O2, and disinfection was by chlorination, but all these steps were on a smaller scale than those at the municipal treatment plants. Cattle, sheep, and goats were slaughtered in the slaughterhouses and the plants treated only domestic wastewater; reclaimed water was reused for irrigation of Shahriār agricultural farm land.

Table 1

Main characteristics of the five WWTPs

        Tertiary Biochemical parameter
 
Water turbiditya
 
  
WWTPs Population served Primary treatment Secondary treatment treatment and disinfection Debit (m3/s) BODc (mg/L) TSSd (mg/L) Influ Efflu Use of treated water 
WWTP1 100,000 Screening and grit removal Activated sludge and A2/Oe Sand filtration followed by chlorination 0.46 <6 <30 High Moderate Discharged into creek 
WWTP2 85,000 Screening and grit removal Conventional activated sludge Chlorination 0.27 <30 <30 High Moderate Discharged to the highway surface water channel 
WWTP3 2,100,000 Screening, grit removal and sedimentation Trickling filter followed by activated sludge Chlorination and UV radiation 5.2 28 28 High Moderate Agriculture irrigation 
SWWTP4 ND Sedimentation Activated sludge and oxidation with O2 Chlorination NDb ND ND High Moderate Agriculture irrigation 
SWWTP5 ND Sedimentation Activated sludge and oxidation with O2 Chlorination ND ND ND High Moderate Agriculture irrigation 
        Tertiary Biochemical parameter
 
Water turbiditya
 
  
WWTPs Population served Primary treatment Secondary treatment treatment and disinfection Debit (m3/s) BODc (mg/L) TSSd (mg/L) Influ Efflu Use of treated water 
WWTP1 100,000 Screening and grit removal Activated sludge and A2/Oe Sand filtration followed by chlorination 0.46 <6 <30 High Moderate Discharged into creek 
WWTP2 85,000 Screening and grit removal Conventional activated sludge Chlorination 0.27 <30 <30 High Moderate Discharged to the highway surface water channel 
WWTP3 2,100,000 Screening, grit removal and sedimentation Trickling filter followed by activated sludge Chlorination and UV radiation 5.2 28 28 High Moderate Agriculture irrigation 
SWWTP4 ND Sedimentation Activated sludge and oxidation with O2 Chlorination NDb ND ND High Moderate Agriculture irrigation 
SWWTP5 ND Sedimentation Activated sludge and oxidation with O2 Chlorination ND ND ND High Moderate Agriculture irrigation 

aLow = <1 NTU, moderate = 1–10 NTU, high = >10 NTU.

bND = no data.

cBOD = biochemical oxygen demand.

dTSS = total suspended solids.

eA2/O = anaerobic/anoxic/oxic.

Sampling

Grab samples of untreated (5 L each) and treated (15 L each) wastewater were collected, monthly from December 2013 to November 2014. Water samples were collected in carboys and transported to the intestinal protozoan laboratory of the faculty of public health at Tehran University of Medical Sciences (TUMS) where they were stored at 4 °C pending analysis. Sample analyses were completed within 7 days. To reduce potential cross-contamination between samples, all carboys used for both raw and treated effluent samples were kept separate. Determinations of sample volume were made according to sample matrix and turbidity in compliance with the Standard Methods for Examination of Water and Wastewater (APHA 2005).

Concentration and purification of Cryptosporidium oocysts and Giardia cysts

The water samples were sieved through a polyester mesh with pore size 297 μm (mesh 50) to remove large particles. Treated and untreated wastewater samples were concentrated by different methods as described below.

Untreated samples were centrifuged at 3,000 × g for 15 min at 4 °C in a 4 × 500 mL capacity swinging-bucket rotor of a refrigerated centrifuge (Beckman, GS-6R Centrifuge). The supernatant fluids were carefully aspirated by a vacuum pump, without disturbing the sediment, and about 100 mL of supernatant was left at the bottom of each canister (Beckman Aerosolve® Cannisters). The residue from each sample was transferred to a 50 mL conical centrifuge tube, and then mixed and centrifuged as before. The water–ether concentration procedure was carried out with 30 mL deionized water and 9 mL diethyl ether. This water–ether concentration procedure was followed by zinc sulfate flotation (sp. gr. 1.3) (Bukhari & Smith 1995). The final pellet was resuspended in 2 mL phosphate-buffered saline (PBS).

Treated samples were pre-filtered with qualitative filter paper to remove large particles such as algae, plant, and other organisms, and immediately submitted to microfiltration using a cellulose-acetate membrane filter (pore size 0.8 μm, 50 mm diameter; Sartorius, Germany). Material adsorbed to the membrane was rinsed with eluting fluid consisting of 0.1% (vol/vol) Tween-80 detergent, 0.1% sodium dodecyl sulfate, NaCl, KH2PO4, Na2HPO4·12H2O and 0.001% (vol/vol) antifoam agent B (Sigma-Aldrich) by scraping the membrane with a smooth-edged plastic loop and subjecting it to centrifugation.

Detection and enumeration of (oo)cysts with direct immunofluorescence assay

An aliquot of 50 μL of pellet was diluted (1:10–1:50) and placed onto a microscope slide with 8 mm diameter wells, air dried, fixed in acetone, and overlaid with 25 μL of fluorescein isothiocyanate (FITC)-conjugated anti-Giardia cysts and anti-Cryptosporidium oocysts monoclonal antibodies (Cellabs Diagnostics, Brookvale, Australia). The slides were incubated at 37 °C in a humid chamber for 30 min. Any excess unbound FITC-antibody was removed by adding 50 mL of PBS to each well (left to stand for 5 min), and then excess PBS was aspirated. A drop (20 μL) of mounting medium (PBS:glycerol, 1:1 v/v) was added to each well, a coverslip was positioned on the top of each drop that was then scanned using microscope fluorescence (Zeiss, Germany) at ×400 magnification.

Cryptosporidium oocysts and Giardia cysts were identified by morphometric criteria including size (oocysts with a mean diameter of 4.9 μm and cysts with a mean diameter of 9.3 μm), shape (spherical), and intensity of immunofluorescent assay staining (bright apple green fluorescence of the oocyst and cyst walls). Positive and negative controls were used as recommended in the Method 1623 (USEPA 1999). The following equation was used to estimate numbers of cysts and oocysts for each sample: 
formula
where α is the number of (oo)cysts multiplied by 106, β is volume of sample in each well (μL), δ is volume of sediment (mL), and Ø is volume of sample (mL).

Cyst viability assays

Cysticidal effectiveness of the treatment plants was evaluated using the eosin exclusion assay (Faubert et al. 1986) and the animal infectivity assay (Garcia et al. 2002).

The eosin exclusion assay was performed with the combination of 0.5 mL of 1:1,000 aqueous eosin solution and 0.5 mL of cyst suspension obtained from effluent samples by standing for 5 min at room temperature. Viable cysts were then counted on a hemocytometer.

The animal infectivity assay was also performed by the method briefly described as follows. Five- to seven-week-old female BALB/c nude immunodeficient mice were provided from the animal-house of the faculty of public health of TUMS. Before inoculation, fecal examination was carried out to ensure that all animals were free from Giardia infections. Eleven mice were divided into three groups of three animals each and one group of two animals in accordance with the inoculum: (i) urban treated effluent; (ii) slaughterhouse treated effluent; (iii) positive control; and (iv) negative control – inoculated with sterile distilled water. To remove bacteria, prior to inoculation, purified cysts were sterilized with 1% NaOCl (60 min at 4 °C). The bleach was washed out with distilled water. Mice (with the exception of the negative control) were inoculated intragastrically by gavage into the animal (with a blunted, smoothed, 26-gauge needle). Each group of mice was housed in a separate micro-isolator cage and subsequent cyst presence was evaluated by collecting feces and immersing in a solution of 2% (wt/vol) potassium dichromate from days 7 to 14 post-inoculation. Samples of feces were then examined by sucrose flotation (sp. gr. 1.18) for the presence of cysts. Mice were anesthetized and necropsied at 2 weeks post-inoculation. The small intestine of each mouse was excised and placed in a Petri dish containing PBS solution. A sample of mucus of the upper small intestine of each mouse was scraped off with a glass slide and stained by Giemsa to test for presence of trophozoites.

Statistical analysis

Data analyses were done with the Student's t-test (paired T-test), nonparametric tests (Wilcoxon-signed ranks test and Mann–Whitney test), and one-way analysis of variance (ANOVA), all used as appropriate. The Kolmogorov–Smirnov test (test of normality) was realized prior to application of the Student's t-test. Statistical analyses were performed with the Statistical Package for the Social Science (SPSS) version 22 (IBM SPSS Inc., Chicago, IL, USA) and statistical difference was considered at P-value < 0.05. The geometric mean concentrations obtained from raw data were also digitalized as spreadsheets using EXCEL software (Microsoft® EXCEL 2010) and statistical graphics were designed. Also evaluated were seasonal slope of standard curves obtained from all plants with cyst and oocyst concentrations in raw wastewater samples and calculations were made for squared correlation coefficient (r2).

RESULTS AND DISCUSSION

Concentrations of Cryptosporidium oocysts and Giardia cysts were determined by immunofluorescence assay (IFA) from influent and effluent wastewater samples from three WWTPs and two SWWTPs. Giardia cysts were found in all influent samples from all plants throughout the year. Mean values were determined in the range of 883.3 ± 416.7 to 3,191.7 ± 1,067.2 cysts per liter, with a maximum of 5,900 and a minimum of 300 cysts per liter (Table 2). Cryptosporidium oocysts were detected in 34 (62.9%) of influent samples from all plants and mean concentrations were determined in the range of 4.8 ± 6.2 to 83.8 ± 77.3 oocysts per liter, with a maximum of 315 and a minimum of 5 oocysts per liter (Table 3). Geometric means of (oo)cysts per liter of raw and treated wastewater samples are shown in Figure 2(a) and 2(b). In influent samples, the mean number of cysts per liter was significantly (P < 0.05) higher than that of oocysts.

Table 2

The mean concentrations of Giardia cysts in raw and treated wastewaters, and cyst removal efficiency of the five treatment plants

WWTP1
 
WWTP2
 
WWTP3
 
SWWTP4
 
SWWTP5
 
  No. cysts/L
 
  No. cysts/L
 
  No. cysts/L
 
  No. cysts/L
 
  No. cysts/L
 
Sa** Influ Efflu Sa Influ Efflu Sa Influ Efflu Sa Influ Efflu Sa Influ Efflu 
E-1 700 80 S-1 2,200 205 G-1 NDa ND M-1 3,400 450 R-1 5,900 600 
E-2 1,900 105 S-2 1,100 95 G-2 ND ND M-2 2,900 750 R-2 2,400 320 
E-3 2,100 155 S-3 1,500 145 G-3 ND ND M-3 1,500 640 R-3 3,600 390 
E-4 1,400 95 S-4 1,600 185 G-4 ND ND M-4 2,200 670 R-4 4,000 410 
E-5 1,100 80 S-5 800 75 G-5 ND ND M-5 2,100 350 R-5 2,100 240 
E-6 800 90 S-6 1,000 95 G-6 ND ND M-6 1,200 400 R-6 3,500 390 
E-7 1,000 85 S-7 1,700 120 G-7 1,400 100 M-7 1,900 550 R-7 3,800 370 
E-8 900 95 S-8 400 35 G-8 300 45 M-8 1,500 350 R-8 2,600 230 
E-9 500 15 S-9 1,100 75 G-9 1,200 115 M-9 1,700 450 R-9 2,200 140 
E-10 1,100 65 S-10 1,000 60 G-10 900 95 M-10 1,400 195 R-10 3,000 410 
E-11 400 40 S-11 1,300 95 G-11 1,000 110 M-11 1,700 145 R-11 2,500 260 
E-12 600 75 S-12 300 55 G-12 500 45 M-12 1,300 350 R-12 2,700 350 
Meanb 1,041.4 ± 529.9 81.7 ± 34.2 Mean 1,166.7 ± 539.9 103.3 ± 52.0 Mean 883.3 ± 416.7 85.0 ± 31.8 Mean 1,900.0 ± 666.1 441.7 ± 184.4 Mean 3,191.7 ± 1,067.2 342.5 ± 117.6 
Pc 0.002  Pc 0.002  Pc 0.028  Pc 0.002  Pc 0.002  
Rd 92.1  91  90.3  76.7  89.2  
WWTP1
 
WWTP2
 
WWTP3
 
SWWTP4
 
SWWTP5
 
  No. cysts/L
 
  No. cysts/L
 
  No. cysts/L
 
  No. cysts/L
 
  No. cysts/L
 
Sa** Influ Efflu Sa Influ Efflu Sa Influ Efflu Sa Influ Efflu Sa Influ Efflu 
E-1 700 80 S-1 2,200 205 G-1 NDa ND M-1 3,400 450 R-1 5,900 600 
E-2 1,900 105 S-2 1,100 95 G-2 ND ND M-2 2,900 750 R-2 2,400 320 
E-3 2,100 155 S-3 1,500 145 G-3 ND ND M-3 1,500 640 R-3 3,600 390 
E-4 1,400 95 S-4 1,600 185 G-4 ND ND M-4 2,200 670 R-4 4,000 410 
E-5 1,100 80 S-5 800 75 G-5 ND ND M-5 2,100 350 R-5 2,100 240 
E-6 800 90 S-6 1,000 95 G-6 ND ND M-6 1,200 400 R-6 3,500 390 
E-7 1,000 85 S-7 1,700 120 G-7 1,400 100 M-7 1,900 550 R-7 3,800 370 
E-8 900 95 S-8 400 35 G-8 300 45 M-8 1,500 350 R-8 2,600 230 
E-9 500 15 S-9 1,100 75 G-9 1,200 115 M-9 1,700 450 R-9 2,200 140 
E-10 1,100 65 S-10 1,000 60 G-10 900 95 M-10 1,400 195 R-10 3,000 410 
E-11 400 40 S-11 1,300 95 G-11 1,000 110 M-11 1,700 145 R-11 2,500 260 
E-12 600 75 S-12 300 55 G-12 500 45 M-12 1,300 350 R-12 2,700 350 
Meanb 1,041.4 ± 529.9 81.7 ± 34.2 Mean 1,166.7 ± 539.9 103.3 ± 52.0 Mean 883.3 ± 416.7 85.0 ± 31.8 Mean 1,900.0 ± 666.1 441.7 ± 184.4 Mean 3,191.7 ± 1,067.2 342.5 ± 117.6 
Pc 0.002  Pc 0.002  Pc 0.028  Pc 0.002  Pc 0.002  
Rd 92.1  91  90.3  76.7  89.2  

**Sample.

aNo data.

bArithmetic mean ± standard deviation.

cWilcoxon-signed ranks test.

dRemoval efficiency (%).

Table 3

The mean concentrations of Cryptosporidium oocysts in raw and treated wastewaters, and oocyst removal efficiency of the five treatment plants

WWTP1
 
WWTP2
 
WWTP3
 
SWWTP4
 
SWWTP5
 
  No. oocysts/L
 
  No. oocysts/L
 
  No. oocysts/L
 
  No. oocysts/L
 
  No. oocysts/L
 
Sa InfluEfflu** Sa Influ Efflu Sa Influ Efflu Sa Influ Efflu Sa Influ Efflu 
E-1 95 S-1 25 15 G-1 NDa ND M-1 105 25 R-1 59 16 
E-2 S-2 22 G-2 ND ND M-2 85 18 R-2 95 62 
E-3 S-3 G-3 ND ND M-3 90 15 R-3 315 25 
E-4 S-4 G-4 ND ND M-4 55 20 R-4 45 29 
E-5 25 S-5 G-5 ND ND M-5 70 15 R-5 80 12 
E-6 S-6 G-6 ND ND M-6 58 10 R-6 120 18 
E-7 S-7 10 G-7 M-7 35 31 R-7 53 24 
E-8 15 S-8 G-8 10 M-8 39 17 R-8 56 21 
E-9 S-9 G-9 M-9 51 22 R-9 75 
E-10 10 S-10 12 G-10 M-10 30 R-10 38 13 
E-11 S-11 G-11 M-11 48 10 R-11 45 22 
E-12 S-12 G-12 15 M-12 34 25 R-12 25 15 
Meanb 12.1 ± 27.3 1.2 ± 2.9 Mean 4.9 ± 9.4 2.5 ± 5 Mean 4.8 ± 6.2 1.7 ± 4.1 Mean 58.3 ± 24.3 18.1 ± 6.9 Mean 83.8 ± 77.3 22.2 ± 13.9 
Pc 0.066 Pc 0.197 Pc 0.285 Pc 0.002 Pc 0.002 
Rd 90.8 48.9 60 69.4 73.6 
WWTP1
 
WWTP2
 
WWTP3
 
SWWTP4
 
SWWTP5
 
  No. oocysts/L
 
  No. oocysts/L
 
  No. oocysts/L
 
  No. oocysts/L
 
  No. oocysts/L
 
Sa InfluEfflu** Sa Influ Efflu Sa Influ Efflu Sa Influ Efflu Sa Influ Efflu 
E-1 95 S-1 25 15 G-1 NDa ND M-1 105 25 R-1 59 16 
E-2 S-2 22 G-2 ND ND M-2 85 18 R-2 95 62 
E-3 S-3 G-3 ND ND M-3 90 15 R-3 315 25 
E-4 S-4 G-4 ND ND M-4 55 20 R-4 45 29 
E-5 25 S-5 G-5 ND ND M-5 70 15 R-5 80 12 
E-6 S-6 G-6 ND ND M-6 58 10 R-6 120 18 
E-7 S-7 10 G-7 M-7 35 31 R-7 53 24 
E-8 15 S-8 G-8 10 M-8 39 17 R-8 56 21 
E-9 S-9 G-9 M-9 51 22 R-9 75 
E-10 10 S-10 12 G-10 M-10 30 R-10 38 13 
E-11 S-11 G-11 M-11 48 10 R-11 45 22 
E-12 S-12 G-12 15 M-12 34 25 R-12 25 15 
Meanb 12.1 ± 27.3 1.2 ± 2.9 Mean 4.9 ± 9.4 2.5 ± 5 Mean 4.8 ± 6.2 1.7 ± 4.1 Mean 58.3 ± 24.3 18.1 ± 6.9 Mean 83.8 ± 77.3 22.2 ± 13.9 
Pc 0.066 Pc 0.197 Pc 0.285 Pc 0.002 Pc 0.002 
Rd 90.8 48.9 60 69.4 73.6 

*Influent.

**Effluent.

aNot data.

bArithmetic mean ± standard deviation.

cWilcoxon-signed ranks test.

dRemoval efficiency (%).

Figure 2

Geometric mean concentrations of Giardia cysts (a) and Cryptosporidium oocysts (b) in the influents of the five plants in spring, summer, autumn, and winter. in, raw wastewater influents; ef, treated wastewater effluents.

Figure 2

Geometric mean concentrations of Giardia cysts (a) and Cryptosporidium oocysts (b) in the influents of the five plants in spring, summer, autumn, and winter. in, raw wastewater influents; ef, treated wastewater effluents.

The mean number of cysts per liter of raw wastewater samples was higher in the autumn–winter period (October–March) (the highest mean: 3,583.3 ± 1,352.7) than in the spring–summer period (April–September) (the highest mean: 2,800 ± 555), although the difference was not statistically significant (P < 0.05) (Table 4).

Table 4

Comparison of Giardia cyst concentrations in effluents of five plants between spring–summer and autumn–winter

  No. Giardia cysts/L raw samples
 
    
WWTPs Spring–Summer Autumn–Winter Mean difference Pa 
WWTP1 750 ± 288.1 1,333.3 ± 575 583.3 0.065 
WWTP2 966.7 ± 535.4 1,366.7 ± 508.6 400 0.394 
WWTP3 883.3 ± 416.7 NDb – – 
SWWTP4 1,583.3 ± 222.9 2,216.7 ± 828 633.3 0.180 
SWWTP5 2,800 ± 555 3,583.3 ± 1,352.7 783.3 0.485 
  No. Giardia cysts/L raw samples
 
    
WWTPs Spring–Summer Autumn–Winter Mean difference Pa 
WWTP1 750 ± 288.1 1,333.3 ± 575 583.3 0.065 
WWTP2 966.7 ± 535.4 1,366.7 ± 508.6 400 0.394 
WWTP3 883.3 ± 416.7 NDb – – 
SWWTP4 1,583.3 ± 222.9 2,216.7 ± 828 633.3 0.180 
SWWTP5 2,800 ± 555 3,583.3 ± 1,352.7 783.3 0.485 

aMann–Whitney test.

bNo data.

In influent samples of all plants, the mean of concentration of oocysts per liter was higher in the autumn–winter period (October–March) (the highest mean: 119 ± 99.6) than in the spring–summer period (April–September) (the highest mean: 48.6 ± 17), and the difference was statistically significant in SWWTP4 (P = 0.004) and SWWTP5 (P = 0.030) (Table 5). The highest number of cysts was detected in autumn and the lowest in summer. The seasonal slope of the standard curves obtained from all plants with cyst and oocyst concentrations in influent samples along with squared correlation coefficient (r2) are shown in Figures 3 and 4.

Table 5

Comparison of Cryptosporidium oocyst concentrations in effluents of five plants between spring–summer and autumn–winter

  No. Cryptosporidium oocysts/L raw samples
 
    
WWTPs Spring–Summer Autumn–Winter Mean difference Pa 
WWTP1 4.2 ± 6.6 20 ± 38.1 15.8 0.703 
WWTP2 2 ± 4.9 7.8 ± 12.2 5.8 0.400 
WWTP3 4.8 ± 6.2 NDb – – 
SWWTP4 39.5 ± 8.3 77.2 ± 19.5 37.7 0.004 
SWWTP5 48.6 ± 17 119 ± 99.6 70.4 0.030 
  No. Cryptosporidium oocysts/L raw samples
 
    
WWTPs Spring–Summer Autumn–Winter Mean difference Pa 
WWTP1 4.2 ± 6.6 20 ± 38.1 15.8 0.703 
WWTP2 2 ± 4.9 7.8 ± 12.2 5.8 0.400 
WWTP3 4.8 ± 6.2 NDb – – 
SWWTP4 39.5 ± 8.3 77.2 ± 19.5 37.7 0.004 
SWWTP5 48.6 ± 17 119 ± 99.6 70.4 0.030 

aMann–Whitney test.

bNo data.

Figure 3

Seasonal slope of the standard curves obtained from all plants with cyst concentrations along with squared correlation coefficient. Giardia cysts were detected in all samples, with the lowest and the highest concentration levels of cysts observed in summer and autumn, respectively. Graph (a) = WWTP1; Graph (b) = WWTP2; Graph (c) = WWTP3; Graph (d) = SWWTP4; Graph (e) = SWWTP5.

Figure 3

Seasonal slope of the standard curves obtained from all plants with cyst concentrations along with squared correlation coefficient. Giardia cysts were detected in all samples, with the lowest and the highest concentration levels of cysts observed in summer and autumn, respectively. Graph (a) = WWTP1; Graph (b) = WWTP2; Graph (c) = WWTP3; Graph (d) = SWWTP4; Graph (e) = SWWTP5.

Figure 4

Seasonal slope of the standard curves obtained from all plants with oocyst concentrations along with squared correlation coefficient. Graph (a) = WWTP1; Graph (b) = WWTP2; Graph (c) = WWTP3; Graph (d) = SWWTP4; Graph (e) = SWWTP5.

Figure 4

Seasonal slope of the standard curves obtained from all plants with oocyst concentrations along with squared correlation coefficient. Graph (a) = WWTP1; Graph (b) = WWTP2; Graph (c) = WWTP3; Graph (d) = SWWTP4; Graph (e) = SWWTP5.

Cryptosporidium oocysts were detected in 55.5% (30/54) of wastewater effluent samples from the five treatment plants and, in positive samples between 5 and 62 oocysts per liter were detected (Table 3). Twenty-six effluent samples from all treatment plants contained higher oocyst concentrations than their respective influent samples (Table 3). Effluent samples from two urban wastewater treatment plants (WWTP2 and WWTP3) contained higher numbers of oocysts than did their respective influents (oocyst removal percentages of 48.8 and 60%, respectively) (Table 3). Effluent samples from two treatment plants (WWTP4 and WWTP5) contained significantly lower (P = 0.002) numbers of oocysts than their respective influent samples (69.4 and 73.6% removal of oocysts, respectively). Giardia cysts were detected in 100% (54) of wastewater effluent samples from the five treatment plants and in positive samples between 15 and 750 cysts per liter were detected (Table 2). Reductions of protozoan (oo)cysts at the three WWTPs were calculated using samples that were positive for both influent and effluent samples. Log10 reduction of cyst concentration (≥90%) was observed in all five plants (t-test; P <0.05). Results determined no statistically significant difference of reduction between the three urban treatment plants for Giardia cysts (ANOVA, P =0.385) and Cryptosporidium oocysts (ANOVA, P =0.548).

The overall removal efficiency of Giardia cysts was 92.1, 91, 90.3, 76.7, and 89.2% for plants WWTP1, WWTP2, WWTP3, SWWTP4, and SWWTP5, respectively (Table 2). Percentages for overall removal efficiency of Cryptosporidium oocysts were 90.8, 48.9, 60, 69.4, and 73.6% for plants WWTP1, WWTP2, WWTP3, SWWTP4, and SWWTP5, respectively (Table 3).

Cyst viability in wastewater effluent samples and the control positive sample was determined by eosin exclusion assay and ranged from 65 to 90%. Giardia cysts recovered from final effluent samples were also administered to groups of mice, as described in Table 6. Each of the three mice in the positive control group and the two mice that received concentrated urban effluent had shed cysts in their feces. Cyst counts in the effluent group were much lower than those in the positive control group. Whereas the positive control group peaked at a mean cyst count of 170, the urban effluent group peaked at a mean of 30 cysts (Table 6). One mouse inoculated with urban effluent and one mouse in the positive control group that shed cysts in its feces each had trophozoites observed in intestinal scrapings (Table 6).

Table 6

Giardia cyst viability assessment

      No. cysts dosed/animal
 
Volume of   Mice with Fecal cyst counts 
Group No. mice per each group Source of cysts Total Viable cystsa (%) inoculated suspension (μl) Mice with cysts in feces trophozoites in intestinal scrapings from mice (cysts per g) 
Final effluent of WWTP ∼1.5 × 102 65 50 ∼30 
ii Final effluent of SWWTP ∼5 × 102 80 50 
iii Control positive ∼5 × 102 90 50 ∼170 
iv Control negative 50 
      No. cysts dosed/animal
 
Volume of   Mice with Fecal cyst counts 
Group No. mice per each group Source of cysts Total Viable cystsa (%) inoculated suspension (μl) Mice with cysts in feces trophozoites in intestinal scrapings from mice (cysts per g) 
Final effluent of WWTP ∼1.5 × 102 65 50 ∼30 
ii Final effluent of SWWTP ∼5 × 102 80 50 
iii Control positive ∼5 × 102 90 50 ∼170 
iv Control negative 50 

aEosin negative.

Since Cryptosporidium oocysts and Giardia cysts have extremely high resistance against chemical disinfectants including chlorine, the presence of (oo)cysts in treated wastewater is a major cause for concern in water reclamation schemes and for discharge into the environment (Carey et al. 2004; Baldursson & Karanis 2011).

The main purpose of the present study was to investigate (oo)cyst removal efficiency of the five WWTPs in Tehran, Iran.

Membrane filtration procedure (scraping and rinsing of membrane) was used to determine concentrations of (oo)cysts in samples of high-turbidity wastewater influent and a centrifugal (water–ether) concentration method for condensation of (oo)cysts in moderate-turbidity wastewater effluent. According to Cantusio Neto et al. (2006), who reported on the use of membrane filtration and centrifugal concentration procedures, the mean recovery percentages of (oo)cysts from source wastewater samples, were 58.8 and 124.0% for cysts, and 26.6 and 65.8% for oocysts, respectively. It should be noted that concentrated wastewater influent is turbid, and an increase in contaminating particulates and a variety of multicellular and unicellular organisms (such as helminthes, bacteria, algae, and protozoa) exerts not only a detrimental effect on (oo)cyst recovery, but also interferes with detection of (oo)cysts. In relation to the raw wastewater samples, which were analyzed by centrifugation (water–ether), primary sedimentation resulted in deposition of all particles and secondary sedimentation (the water–ether concentration) resulted in less turbid samples which were more easily analyzed under a microscopic. The use of a water–ether concentration procedure before flotation greatly improved (oo)cyst clarification prior to microscopic detection, moreover this method facilitated analysis of very turbid samples such as slaughterhouse waste. An approved standard method for Giardia cyst and Cryptosporidium oocyst detection in aquatic environmental samples is described in the USEPA 1623 protocol, and most existing methods are based on the protocol (Guillot & Loret 2009). These methods generally rely on concentration, purification, and detection steps, and recoveries of <1 to 61% have been reported, dependent on method, seeding level, and turbidity of the sample (Rimhanen-Finne 2006).

The present study was the first attempt to detect naturally occurring Cryptosporidium oocysts and Giardia cysts in wastewater samples in Iran.

The concentration of Giardia cysts in wastewater samples (300–5,900 cysts per liter in influent, 15–750 cysts per liter in effluent) was within the range of those reported in previous studies (Lim et al. 2007; Bertrand & Schwartzbrod 2007; Castro-Hermida et al. 2008; Ben Ayed-Khouja et al. 2010; Kitajima et al. 2014).

Similarly, the concentration of Giardia cysts in the wastewater samples (5–315 cysts per liter in influent, 5–62 cysts per liter in effluent) was similar to those observed in previous studies (Lim et al. 2007; Castro-Hermida et al. 2008; Kitajima et al. 2014).

Concentrations of Giardia in influent samples were always higher than those of Cryptosporidium and reduction efficiency of Giardia was also higher than that of Cryptosporidium. Similar results have been reported in previous studies (Ottoson et al. 2006; Lim et al. 2007; Castro-Hermida et al. 2008).

These findings suggest that Giardia was more widely spread in humans, and reports of clinical observations have indicated that giardiasis was more frequently reported than cryptosporidiosis; this hypothesis has been assumed in epidemiological data published in Iran (Taghipour et al. 2011; Abbasian et al. 2012). In other words, there is a correlation between concentrations of parasites in wastewater and prevalence of parasitosis in communities. Furthermore, monitoring levels of these waterborne pathogens in wastewater can be useful for surveillance of prevalence of giardiosis and cryptosporidiosis in the community to give a more accurate perspective of endemic parasitosis than can be achieved by epidemiological data; as this type of monitoring takes into account all of the clinically asymptomatic carriers and laboratory misdiagnosis and undiagnosed cases that ordinarily escape detection in epidemiological surveys (CDC 2010a, b).

In the present study, the maximum number of (oo)cysts in the influent samples was lower than that reported by Cacciò et al. (2003), and it seems that the number of (oo)cysts detected in positive samples is an under-estimation; however, proper evaluation of recovery efficiency from methods used for isolation and enumeration of (oo)cysts is seldom quoted.

In fact, interpretation and comparison of data are difficult because of differences such as rate of infection within populations and size of populations studied, wastewater type (domestic versus urban), type of secondary treatment system, time of sampling, sample volume and type (grab or large volume), and the different concentration techniques that are used for (oo)cyst detection.

In the present study, evaluations of (oo)cyst concentrations were different throughout the study period with the highest number of (oo)cysts detected in autumn and winter. Previous studies report that concentrations of Giardia cysts in environmental waters increased in colder and wetter months (Cacciò et al. 2003; Oda et al. 2005). A number of studies have reported occurrences of Cryptosporidium and Giardia in wastewater (Robertson et al. 2000, 2006; Bonadonna et al. 2002; Cacciò et al. 2003; Harwood et al. 2005; Oda et al. 2005; Hashimoto et al. 2006; McCuin & Clancy 2006; Ottoson et al. 2006; Bertrand & Schwartzbrod 2007; Gallas-Lindemann et al. 2013), but there remains limited quantitative information on the removal of these (oo)cysts by wastewater treatment (Table 7). Documented evidence indicates that (oo)cysts can pass through conventional treatment systems with reported efficiencies of removal varying from a zero to 2 log reduction dependent upon degree of secondary treatment and concentration of suspended solids (Lim et al. 2007; Jiménez et al. 2010).

Table 7

Occurrence of Cryptosporidium and Giardia ((oo)cysts per liter) in WWTP influents and effluents in our study in comparison with other countries

Study area TSa Influent cyst con* Effluent cyst con % or log10 removal Influent oocyst con Effluent oocyst con* % or log10 removal Method (Oo)cysts viability assay The study conducted by 
UK Settlement and activated sludge 948 ± 361; 923 ± 1,082; 7,600 ± 6,079 15 ± 15; 15 ± 24; 7 ± 16 99 ± 4% 20 ± 28; 19 ± 37; 113 ± 125 5 ± 8; 2 ± 7; n.d. 91 ± 31% Ec; SF; C DAPI & PI Robertson et al. (2000)  
UK Settlement and activated sludge 1,242 ± 1,226; 274 ± 283; 2,809 ± 1,455 62 ± 138; 523 ± 1,126; 1,045 ± 2,202 69 ± 26% 12 ± 19; 9 ± 13; 18 ± 60 22 ± 28; 12 ± 19; 12 ± 30 15 ± 33% Ec; SF; C DAPI & PI Robertson et al. (2000)  
UK Activated sludge 668 ± 986; 843 ± 1,294 63 ± 54; 27 ± 26 66 ± 35% 668 ± 986; 843 ± 1,294 16 ± 28; 8 ± 8 28 ± 34% Ec; SF DAPI & PI Robertson et al. (2000)  
UK Settlement 1,270 ± 782; 2,284 ± 2,486; 16,429 ± 9,408 628 ± 297; 812 ± 896; 8,386 ± 4,262 47 ± 34% 6 ± 10; 43 ± 44; 143 ± 62 16 ± 16; 46 ± 39; 43 ± 79 30 ± 40% Ec; SF; C DAPI & PI Robertson et al. (2000)  
UK Settlement and trickling filters 963 ± 701; 1,517 ± 1,564; 15,486 ± 4,317 182 ± 160; 80 ± 101; 2,029 ± 1,207 85 ± 15% 7 ± 14; n.d.; n.d. 7 ± 8; 25 ± 32; 343 ± 395 5 ± 35% Ec; SF; C DAPI & PI Robertson et al. (2000)  
UK Settlement and trickling filters and sand filtration 3,383 ± 3,516; 1,446 ± 1,541; 2,809 ± 1,455 237 ± 179; 669 ± 858; 1,045 ± 2,202 74 ± 30% 4 ± 7; 6 ± 7; 111 ± 127 11 ± 15; 6 ± 7; 22 ± 44 38 ± 38% Ec; SF; C DAPI & PI Robertson et al. (2000)  
Italy Oxidation with O2 and sedimentation mean 2,100–42,000 n.f. 94.5% 2.5–40 n.f. – ADM & IFA & PCR – Caccio et al. (2003)  
Italy Activated sludge and sedimentation mean 2,100–42,000 n.f. 87.0% n.f. n.f. – ADM & IFA & PCR – Caccio et al. (2003)  
Italy Activated sludge and sedimentation mean 2,100–42,000 n.f. 96.0% n.f. n.f. – ADM & IFA & PCR – Caccio et al. (2003)  
Italy Activated sludge and sedimentation and filtration (60-μm pore) mean 2,100–42,000 n.f. 98.4% 277 n.f. – ADM & IFA & PCR – Caccio et al. (2003)  
Spain Activated sludge and sedimentation – – – mean 125.87 – – MF & IMS & IFA DAPI/PI Montemayor et al. (2005)  
Spain Physical–chemical precipitation – – – mean 126.94 – – MF & IMS & IFA DAPI/PI Montemayor et al. (2005)  
Spain Activated sludge and sedimentation and sand filtration – – – mean 103.71 mean 0.14 – MF & IMS & IFA DAPI/PI Montemayor et al. (2005)  
Spain Activated sludge and sedimentation and lagooning and wet land – – – mean 139.10 mean 0.22 – MF & IMS & IFA DAPI/PI Montemayor et al. (2005)  
Spain Activated sludge and sedimentation and sand filtration – – – mean 130.29 mean 0.31 – MF & IMS & IFA DAPI/PI Montemayor et al. (2005)  
Brazil Activated sludge and UV radiation mean 1,00,000 ± 8.7 mean 1,100 98.9% mean 60,000 ± 2.8 160 (one sample) 99.7% MF & IFA Animal infectivity assay Cantusio Neto et al. (2006)  
Malaysia Oxidation with O2 and sedimentation (extended aeration) 18–5,240 1–500 average 96% 1–80 20–40 average 73% F & IFA DAPI Lim et al. (2007)  
Malaysia Oxidation with O2 and sedimentation (aerated lagoon) 55–8,480 28–1,462 average 92% 40–80 20–80 average 33% F & IFA DAPI Lim et al. (2007)  
Spain Activated sludge and sedimentation 3,450–10,000 227–3,600 – 16–960 20–1,120 – MF & IMS & IFA DAPI Castro-Hermida et al. (2008)  
Spain Oxidation with O2 and sedimentation 1,440–4,000 200–6,000 – 3–400 20–200 – MF & IMS & IFA DAPI Castro-Hermida et al. (2008)  
Spain Activated sludge and sedimentation 39–587 13–1,184 – 1–20 2–260 – MF & IMS & IFA DAPI Castro-Hermida et al. (2008)  
Spain Activated sludge and sedimentation 3–2,667 2–244 – 3–23 2–5 – MF & IMS & IFA DAPI Castro-Hermida et al. (2008)  
Spain Activated sludge and sedimentation 240–8,000 194–1,100 – 2–49 1–50 – MF & IMS & IFA DAPI Castro-Hermida et al. (2008)  
Spain Activated sludge and sedimentation 2,400–3,920 384–2,000 – 4–178 2–94 – MF & IMS & IFA DAPI Castro-Hermida et al. (2008)  
Spain Activated sludge and sedimentation 2,240–14,400 34–1,867 – 1–248 2–35 – MF & IMS & IFA DAPI Castro-Hermida et al. (2008)  
Spain Biological aerated filters 4–164 24–578 – 1–46 1–16 – MF & IMS & IFA DAPI Castro-Hermida et al. (2008)  
Spain Oxidation with O2 800–9,000 3–880 – 4–8 1–8 – MF & IMS & IFA DAPI Castro-Hermida et al. (2008)  
Spain Oxidation with O2 85–10,000 50–1,440 – 1–160 1–20 – MF & IMS & IFA DAPI Castro-Hermida et al. (2008)  
Spain Oxidation with O2 320–4,000 400–973 – 1–380 1–54 – MF & IMS & IFA DAPI Castro-Hermida et al. (2008)  
Spain Oxidation with O2 2–448 5–4,360 – 1–112 1–120 –; – MF & IMS & IFA DAPI Castro-Hermida et al. (2008)  
Tunisia Oxidation channel and drying beds 80–320; 28–60 0; n.d. –; – –; 8–9 –; 0 –; – MBM; & IMS & IFA – Ben Ayed-Khouja et al. (2010)  
Tunisia Oxidation channel and dehydration 260; 37 n.d.; n.d. –; – –; 5 –; 0 –; – MBM; & IMS & IFA – Ben Ayed-Khouja et al. (2010)  
Tunisia Activated sludge and dehydration and drying beds 66–220; 0 0–n.d.; 1 –; – –; 8 –; 0 –; – MBM; & IMS & IFA – Ben Ayed-Khouja et al. (2010)  
Tunisia Aerated lagoon n.d.; 106 n.d.; 2 –; – –; 21 –; – –; – MBM; & IMS & IFA – Ben Ayed-Khouja et al. (2010)  
Tunisia Stabilization pond n.d.; n.d. 0; 4 –; – –; n.d. –; n.d. –; – MBM; & IMS & IFA – Ben Ayed-Khouja et al. (2010)  
Tunisia Oxidation channel and anaerobic digestion 160; 18 0; 2 –; – –; 2 –; 0 –; – MBM; & IMS & IFA – Ben Ayed-Khouja et al. (2010)  
Iran Conventional activated sludge mean 10.77 mean 0.35 99% – – – MBM – Sharafi et al. (2012)  
Iran Stabilization ponds mean 7.6 ≥99% – – – MBM – Sharafi et al. (2012)  
Iran Extended aeration activated sludge mean 14.44 mean 0.24 99% – – – MBM – Sharafi et al. (2012)  
Iran Constructed wetland mean 6.85 ≥99% – – – MBM – Sharafi et al. (2012)  
Iran Extended aeration activated sludge mean 15.55 mean 0.2 99% – – – MBM – Sharafi et al. (2012)  
Iran Stabilization ponds mean 9.11 ≥99% – – – MBM – Sharafi et al. (2012)  
USA Activated sludge mean 4,800 mean 33 2.08 ± 0.44 log10 reduction mean 74 mean 12 0.71 ± 0.20 log10 MF & IMS & IFA – Kitajima et al. (2014)  
USA Trickling filter mean 6,400 mean 190 1.52 ± 0.62 log10 reduction mean 100 mean 13 0.81 ± 0.22 log10 MF & IMS & IFA – Kitajima et al. (2014)  
Iran Activated sludge – A2/O and sand filtration Meanb 928.16 mean 72.48 92.1% meanc 12.1 ± 27.3 mean 1.2 ± 2.9 90.8% MF & F & CC & IFA EEA & AIA Our study 
Iran Conventional activated sludge mean 1,021.69 mean 91.99 91% mean 4.9 ± 9.4 mean 2.5 ± 5 48.9% MF & F & CC & IFA EEA & AIA Our study 
Iran Activated sludge and trickling filter mean 780.92 mean 79.01 90.3% mean 4.8 ± 6.2 mean 1.7 ± 4.1 60% MF & F & CC & IFA EEA & AIA Our study 
Iran Activated sludge mean 1,809.11 mean 401.15 76.7% mean 58.3 ± 24.3 mean 18.1 ± 6.9 69.4% MF & F & CC & IFA EEA & AIA Our study 
Iran Activated sludge mean 3,054.52 mean 322.74 89.2% mean 83.8 ± 77.3 mean 22.2 ± 13.9 73.6% MF & F & CC & IFA EEA & AIA Our study 
Study area TSa Influent cyst con* Effluent cyst con % or log10 removal Influent oocyst con Effluent oocyst con* % or log10 removal Method (Oo)cysts viability assay The study conducted by 
UK Settlement and activated sludge 948 ± 361; 923 ± 1,082; 7,600 ± 6,079 15 ± 15; 15 ± 24; 7 ± 16 99 ± 4% 20 ± 28; 19 ± 37; 113 ± 125 5 ± 8; 2 ± 7; n.d. 91 ± 31% Ec; SF; C DAPI & PI Robertson et al. (2000)  
UK Settlement and activated sludge 1,242 ± 1,226; 274 ± 283; 2,809 ± 1,455 62 ± 138; 523 ± 1,126; 1,045 ± 2,202 69 ± 26% 12 ± 19; 9 ± 13; 18 ± 60 22 ± 28; 12 ± 19; 12 ± 30 15 ± 33% Ec; SF; C DAPI & PI Robertson et al. (2000)  
UK Activated sludge 668 ± 986; 843 ± 1,294 63 ± 54; 27 ± 26 66 ± 35% 668 ± 986; 843 ± 1,294 16 ± 28; 8 ± 8 28 ± 34% Ec; SF DAPI & PI Robertson et al. (2000)  
UK Settlement 1,270 ± 782; 2,284 ± 2,486; 16,429 ± 9,408 628 ± 297; 812 ± 896; 8,386 ± 4,262 47 ± 34% 6 ± 10; 43 ± 44; 143 ± 62 16 ± 16; 46 ± 39; 43 ± 79 30 ± 40% Ec; SF; C DAPI & PI Robertson et al. (2000)  
UK Settlement and trickling filters 963 ± 701; 1,517 ± 1,564; 15,486 ± 4,317 182 ± 160; 80 ± 101; 2,029 ± 1,207 85 ± 15% 7 ± 14; n.d.; n.d. 7 ± 8; 25 ± 32; 343 ± 395 5 ± 35% Ec; SF; C DAPI & PI Robertson et al. (2000)  
UK Settlement and trickling filters and sand filtration 3,383 ± 3,516; 1,446 ± 1,541; 2,809 ± 1,455 237 ± 179; 669 ± 858; 1,045 ± 2,202 74 ± 30% 4 ± 7; 6 ± 7; 111 ± 127 11 ± 15; 6 ± 7; 22 ± 44 38 ± 38% Ec; SF; C DAPI & PI Robertson et al. (2000)  
Italy Oxidation with O2 and sedimentation mean 2,100–42,000 n.f. 94.5% 2.5–40 n.f. – ADM & IFA & PCR – Caccio et al. (2003)  
Italy Activated sludge and sedimentation mean 2,100–42,000 n.f. 87.0% n.f. n.f. – ADM & IFA & PCR – Caccio et al. (2003)  
Italy Activated sludge and sedimentation mean 2,100–42,000 n.f. 96.0% n.f. n.f. – ADM & IFA & PCR – Caccio et al. (2003)  
Italy Activated sludge and sedimentation and filtration (60-μm pore) mean 2,100–42,000 n.f. 98.4% 277 n.f. – ADM & IFA & PCR – Caccio et al. (2003)  
Spain Activated sludge and sedimentation – – – mean 125.87 – – MF & IMS & IFA DAPI/PI Montemayor et al. (2005)  
Spain Physical–chemical precipitation – – – mean 126.94 – – MF & IMS & IFA DAPI/PI Montemayor et al. (2005)  
Spain Activated sludge and sedimentation and sand filtration – – – mean 103.71 mean 0.14 – MF & IMS & IFA DAPI/PI Montemayor et al. (2005)  
Spain Activated sludge and sedimentation and lagooning and wet land – – – mean 139.10 mean 0.22 – MF & IMS & IFA DAPI/PI Montemayor et al. (2005)  
Spain Activated sludge and sedimentation and sand filtration – – – mean 130.29 mean 0.31 – MF & IMS & IFA DAPI/PI Montemayor et al. (2005)  
Brazil Activated sludge and UV radiation mean 1,00,000 ± 8.7 mean 1,100 98.9% mean 60,000 ± 2.8 160 (one sample) 99.7% MF & IFA Animal infectivity assay Cantusio Neto et al. (2006)  
Malaysia Oxidation with O2 and sedimentation (extended aeration) 18–5,240 1–500 average 96% 1–80 20–40 average 73% F & IFA DAPI Lim et al. (2007)  
Malaysia Oxidation with O2 and sedimentation (aerated lagoon) 55–8,480 28–1,462 average 92% 40–80 20–80 average 33% F & IFA DAPI Lim et al. (2007)  
Spain Activated sludge and sedimentation 3,450–10,000 227–3,600 – 16–960 20–1,120 – MF & IMS & IFA DAPI Castro-Hermida et al. (2008)  
Spain Oxidation with O2 and sedimentation 1,440–4,000 200–6,000 – 3–400 20–200 – MF & IMS & IFA DAPI Castro-Hermida et al. (2008)  
Spain Activated sludge and sedimentation 39–587 13–1,184 – 1–20 2–260 – MF & IMS & IFA DAPI Castro-Hermida et al. (2008)  
Spain Activated sludge and sedimentation 3–2,667 2–244 – 3–23 2–5 – MF & IMS & IFA DAPI Castro-Hermida et al. (2008)  
Spain Activated sludge and sedimentation 240–8,000 194–1,100 – 2–49 1–50 – MF & IMS & IFA DAPI Castro-Hermida et al. (2008)  
Spain Activated sludge and sedimentation 2,400–3,920 384–2,000 – 4–178 2–94 – MF & IMS & IFA DAPI Castro-Hermida et al. (2008)  
Spain Activated sludge and sedimentation 2,240–14,400 34–1,867 – 1–248 2–35 – MF & IMS & IFA DAPI Castro-Hermida et al. (2008)  
Spain Biological aerated filters 4–164 24–578 – 1–46 1–16 – MF & IMS & IFA DAPI Castro-Hermida et al. (2008)  
Spain Oxidation with O2 800–9,000 3–880 – 4–8 1–8 – MF & IMS & IFA DAPI Castro-Hermida et al. (2008)  
Spain Oxidation with O2 85–10,000 50–1,440 – 1–160 1–20 – MF & IMS & IFA DAPI Castro-Hermida et al. (2008)  
Spain Oxidation with O2 320–4,000 400–973 – 1–380 1–54 – MF & IMS & IFA DAPI Castro-Hermida et al. (2008)  
Spain Oxidation with O2 2–448 5–4,360 – 1–112 1–120 –; – MF & IMS & IFA DAPI Castro-Hermida et al. (2008)  
Tunisia Oxidation channel and drying beds 80–320; 28–60 0; n.d. –; – –; 8–9 –; 0 –; – MBM; & IMS & IFA – Ben Ayed-Khouja et al. (2010)  
Tunisia Oxidation channel and dehydration 260; 37 n.d.; n.d. –; – –; 5 –; 0 –; – MBM; & IMS & IFA – Ben Ayed-Khouja et al. (2010)  
Tunisia Activated sludge and dehydration and drying beds 66–220; 0 0–n.d.; 1 –; – –; 8 –; 0 –; – MBM; & IMS & IFA – Ben Ayed-Khouja et al. (2010)  
Tunisia Aerated lagoon n.d.; 106 n.d.; 2 –; – –; 21 –; – –; – MBM; & IMS & IFA – Ben Ayed-Khouja et al. (2010)  
Tunisia Stabilization pond n.d.; n.d. 0; 4 –; – –; n.d. –; n.d. –; – MBM; & IMS & IFA – Ben Ayed-Khouja et al. (2010)  
Tunisia Oxidation channel and anaerobic digestion 160; 18 0; 2 –; – –; 2 –; 0 –; – MBM; & IMS & IFA – Ben Ayed-Khouja et al. (2010)  
Iran Conventional activated sludge mean 10.77 mean 0.35 99% – – – MBM – Sharafi et al. (2012)  
Iran Stabilization ponds mean 7.6 ≥99% – – – MBM – Sharafi et al. (2012)  
Iran Extended aeration activated sludge mean 14.44 mean 0.24 99% – – – MBM – Sharafi et al. (2012)  
Iran Constructed wetland mean 6.85 ≥99% – – – MBM – Sharafi et al. (2012)  
Iran Extended aeration activated sludge mean 15.55 mean 0.2 99% – – – MBM – Sharafi et al. (2012)  
Iran Stabilization ponds mean 9.11 ≥99% – – – MBM – Sharafi et al. (2012)  
USA Activated sludge mean 4,800 mean 33 2.08 ± 0.44 log10 reduction mean 74 mean 12 0.71 ± 0.20 log10 MF & IMS & IFA – Kitajima et al. (2014)  
USA Trickling filter mean 6,400 mean 190 1.52 ± 0.62 log10 reduction mean 100 mean 13 0.81 ± 0.22 log10 MF & IMS & IFA – Kitajima et al. (2014)  
Iran Activated sludge – A2/O and sand filtration Meanb 928.16 mean 72.48 92.1% meanc 12.1 ± 27.3 mean 1.2 ± 2.9 90.8% MF & F & CC & IFA EEA & AIA Our study 
Iran Conventional activated sludge mean 1,021.69 mean 91.99 91% mean 4.9 ± 9.4 mean 2.5 ± 5 48.9% MF & F & CC & IFA EEA & AIA Our study 
Iran Activated sludge and trickling filter mean 780.92 mean 79.01 90.3% mean 4.8 ± 6.2 mean 1.7 ± 4.1 60% MF & F & CC & IFA EEA & AIA Our study 
Iran Activated sludge mean 1,809.11 mean 401.15 76.7% mean 58.3 ± 24.3 mean 18.1 ± 6.9 69.4% MF & F & CC & IFA EEA & AIA Our study 
Iran Activated sludge mean 3,054.52 mean 322.74 89.2% mean 83.8 ± 77.3 mean 22.2 ± 13.9 73.6% MF & F & CC & IFA EEA & AIA Our study 

*Concentration.

aTreatment processes of WWTPs.

bGeometric mean.

cArithmetic mean.

DAPI = 4′,6-diamidino-2-phenylindole; PI = propidium iodide; Ec = ether concentration; SF = sucrose flotation; C = centrifugation; IFA = immunofluorescent antibody; ADM = acetone dissolution method; PCR = polymerase chain reaction; n.f. = not found; MF = membrane filtration; IMS = immunomagnetic separation; F = flotation; MBM = modified Bailenger method; CC = centrifugal-(water–ether) concentration; EEA = eosin exclusion assay; AIA = animal infectivity assay.

One of the main objectives of this present study was to compare the removal efficiency of (oo)cysts at WWTPs from different biological wastewater treatment processes, namely, activated sludge-A2O and sand filtration (WWTP1), conventional activated sludge (WWTP2), and activated sludge with a trickling filter (WWTP3). In these plants, (oo)cysts were physically removed from wastewater by conventional separation processes including bacterial flocculation and settling (activated sludge), and flocculation and settling prior to filtration (trickling filtration process). No statistically significant difference of (oo)cyst removal reduction was observed between the three plants. A number of comparative studies have concluded that the activated sludge treatment process is more effective at removing Giardia than trickling filtration (Wiandt et al. 2000; Kitajima et al. 2014). In contrast, Robertson et al. (2000) found no significant difference in the removal of Giardia and Cryptosporidium between the three tested plants.

In this study, despite a substantial reduction in (oo)cyst concentration, high (oo)cyst concentrations were detected in final effluent samples from all treatment plants, and a proportion of Giardia cysts and Cryptosporidium oocysts reached the environment by means of discharge of finished waters into creeks, surface water channels, and in agricultural irrigation purposes. Accordingly, the second aim of this study was to evaluate whether or not cysts exposed to treatment processes were capable of retaining their infectivity, given the risks to public health. In microbiological studies on water and wastewater effluent, determination of (oo)cyst concentrations without viability or infectivity assessment may significantly overestimate the potential health risks associated with protozoan (oo)cysts in final wastewater effluent samples (Garcia et al. 2002). The best method to assess the (oo)cyst affectivity of treatment processes is in vivo testing using a suitable animal model or cell culture (for Cryptosporidium). In fact, in vitro excystation using enzymatic capabilities of the oocyst to open up exposure to trypsin at 37 °C, or fluorogenic vital dyes such as 4,6-diamidino-2-phenylindole (DAPI), and propidium iodide (PI) have a percentage error and therefore cannot be relied upon for accurate estimation of post-treatment (oo)cyst infectivity (Quintero-Betancourt & Rose 2004). Alternative physical disinfectants such as ultra violet (UV) irradiation have been shown to affect DNA such that, while membranes and enzymes seem to be intact, the organism is no longer capable of reproducing (Morita et al. 2002). Huffman et al. (2000) reported that vital dyes overestimated Cryptosporidium infectivity, while predictions using the Focus Detection Method-Most Probable Number cell culture method were comparable to the use of animal infectivity.

In this study, cyst viability was first assessed using the eosin exclusion assay, followed by animal infectivity assay to overcome the biases due to the overestimation of the viability of Giardia cysts by the eosin exclusion procedure (Thiriat et al. 1998). The presence of viable and infectious cysts in final effluent samples was confirmed by the results of eosin exclusion and animal infectivity assays in this study. One mouse inoculated with urban effluent and one mouse in the positive control group, each shed cysts in its feces and trophozoites were observed in their intestinal scrapings, thus demonstrating the presence of infectious cysts in the final effluent sample. Animals infected with urban effluent cysts had low cyst counts in feces and this may be attributed to difference in potential viability of cysts (65%) compared with the positive control cysts (90%), a possible difference in cyst concentrations between the two groups that were inoculated, or a combination of both factors. In group two, the failure to infect mice with effluent cysts could stem from the fact that the virulence of cysts (anthropozoonotic strains, e.g., assemblages A and B, or specific strains for their hosts, e.g., assemblage E) in animal effluent samples was not appropriate to infect the laboratory animals used in the tests.

Cryptosporidium oocysts and Giardia cysts are known to be resistant to chlorine at concentrations typically applied for water treatment. Free chlorine levels of up to 16,000 mg per liter are required to completely inactivate oocysts, and chlorination with dosages normally used for wastewater disinfection of secondary effluents (2–15 mg per liter) and filtered secondary effluent (1–5 mg per liter) is ineffective for removing (oo)cysts (Chauret et al. 2001). The (oo)cysts have also been detected in wastewater final-effluent after chlorination, UV radiation and ozonation (ranging from 0.26–7 oocysts per liter to 0.3–104 cysts per liter), although, evaluation of infectivity of (oo)cysts was not determined (Liberti et al. 2000; Wiandt et al. 2000). In Brazil, Cantusio Neto et al. (2006) reported that trophozoites were successfully excysted in mice after inoculation of cysts found in wastewater final-effluent samples (post-UV treatment).

It should be noted that (oo)cysts readily attach to the biological particles and this attachment to particles will influence not only sedimentation of (oo)cysts in the sludge, but probably affects survival of (oo)cysts and their removal during wastewater treatment by sand filtration and disinfection processes (chlorination and UV radiation). (Oo)cysts exhibit variation in their zeta potential when suspended in water-based solutions of varying conductivity, pH, and dissolved organic carbon concentration. Cryptosporidium oocysts, when placed in the presence of dissolved compounds from natural organic matters, exhibit an absolute increase of negative charges and of hydrophobicity, possibly due to the adsorption of clay and humic and fulvic acids onto their surfaces. This may enhance transport rather than parasite sedimentation (Dumètre et al. 2012). However, humic acids, phenolic compounds, and lignin sulfonates, as well as chromium, cobalt, copper, and nickel can decrease UV transmission (USEPA 1999).

There are only a few studies that have focused on assessment of Giardia cyst removal efficiency of urban WWTPs in Iran (Sharafi et al. 2012). To date, no study has described the presence of Cryptosporidium oocysts in untreated and treated wastewaters in Iran, and this work provides the first data on distributions of Giardia and Cryptosporidium in wastewater influent and effluent samples, (oo)cyst removal efficiency of treatment plants, and cysticidal effectiveness of treatment processes using an animal model. In the study area on the outskirts of Tehran, in addition to slaughterhouses, there were many intensive farming and ranching operations in the region and the use of fertilizers of animal origin by farmers may also have contributed to the contamination of surface waters and crops and herbs. Further studies can contribute to this study by analyzing Giardia duodenalis and Cryptosporidium spp. at the genotype level, simultaneously from environmental and fecal samples, and this could lead to better evaluation of sources of fecal contamination in environmental samples. The presence of Cryptosporidium oocysts and Giardia cysts in treated wastewater can present a serious public health concern. Contamination wastewater released into surface and groundwater sources could increase the risk of human giardiasis and cryptosporidiosis through consumption of vegetables and use of water.

CONCLUSION

These results have demonstrated that Giardia and Cryptosporidium were prevalent in the study area and this highlights the importance of treating wastewater effluent from domestic and urban treatment plants to control pathogenic protozoans. The tested wastewater treatment processes (activated sludge) were determined as having limited efficiency in terms of removing (oo)cysts, so it is important that wastewater treatment authorities reconsider the relevance of protozoa contamination levels in wastewater and that appropriate countermeasures are developed with suitable regulations on treatment processes such as membrane-based technologies (e.g., ultrafiltration) that not only establish acceptable levels of reduction of (oo)cysts but that also improve the levels of acceptable quality of recycled wastewaters to avoid chemical disinfection and consequent possible formation of toxic by-products. These improvements can also include consideration of supplementary inline physical disinfection systems known to inactivate Cryptosporidium oocysts and Giardia cysts (such as UV radiation or ozonation) with increased exposure time to pass radiation through the (oo)cyst wall and to increase the impact of physical disinfectants on (oo)cysts.

ACKNOWLEDGEMENTS

We thank Dr Hamidreza Tashayoi and Mr Rouhi at the Iran Water and Wastewater Treatment Company for their administrative cooperation. We are also grateful to Dr A. R. Meamar for providing Giardia cysts. This study received financial support from research grant 92–02–160–23616, entitled ‘Assessment of phenotypic and genotypic of Giardia and Cryptosporidium in the influent and in the effluent of human and domestic animal wastewater treatment plants of Tehran’ of the Institute for Environmental Research (IER), Tehran University of Medical Sciences, and Center for Research of Endemic Parasites of Iran (CREPI), Tehran University of Medical Sciences. The authors declare that they have no competing interests.

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