Assessment of Cryptosporidium Removal from ... - Springer Link

Water Air Soil Pollut (2007) 179:207–215 DOI 10.1007/s11270-006-9225-8

Assessment of Cryptosporidium Removal from Domestic Wastewater Via Constructed Wetland Systems Effat A. Morsy & Ahmad Z. Al-Herrawy & Mohamed A. Ali

Received: 6 March 2006 / Accepted: 21 June 2006 / Published online: 12 September 2006 # Springer Science + Business Media B.V. 2006

Abstract Constructed wetlands have been recognized as offering a removal treatment option for high concentrations removal of chemical and biological contaminants in domestic wastewater. The enteric protozoan parasite Cryptosporidium is considered one of the highly resistant to treatment and highly infectious organisms to humans and animals. Moreover, some species of Cryptosporidium are known to have a zoonotic nature. In this investigation a pilot scale for domestic wastewater treatment system was used, consisting of the following steps in series: (1) up-flow anaerobic sludge blanket (UASB) reactor, (2) free water surface (FWS) wetland unit, and (3) sub-surface flow (SSF) wetland unit. This treatment system was fed with domestic wastewater to assess its efficiency in removing Cryptosporidium oocysts. The obtained Cryptosporidium oocysts were detected and enumerated by two different staining techniques ‘acid fast trichrome (AFT) and modified Ziehl Neelsen (MZN) stains_. Polymerase chain reaction (PCR) technique was also used to detect Cryptosporidium DNA in wastewater samples. Results revealed that anaerobic treatment (using UASB reactor) could remove about 53.1% of Cryptosporidium oocysts present in raw E. A. Morsy (*) : A. Z. Al-Herrawy : M. A. Ali Water Pollution Research Department, Environmental Research Division National Research Centre, Tahrir St., Cairo, Dokki P.C. 12311, Egypt e-mail: [email protected]

wastewater. The in-series connection between the two wetland units allowed complete elimination of Cryptosporidium oocysts as the first (FWS) wetland unit removed 95.9% of the oocysts present in anaerobically treated wastewater and the remaining portion of oocysts was completely removed by the second (SSF) wetland unit. Cryptosporidium oocysts were detected in 95.8% of raw wastewater samples with a mean count of 43.8 oocysts/l when AFT stain was used while they were detected in only 87.5% of raw wastewater samples with a mean count of 35.6 oocysts/l when MZN stain was used. Polymerase chain reaction (PCR) technique was able to detect Cryptosporidium DNA in only 45.8% of raw wastewater samples. Positive PCR results were only achieved in wastewater samples containing 52 oocysts or more per liter. Keywords constructed wetlands . Cryptosporidium . domestic wastewater . PCR technique

1 Introduction At the global level, there has been a growing shortage of freshwater reserves, mainly those of good quality, as a result of increasing in human consumption and decreasing annual rain fall. To address this problem, treated wastewater with a low level of chemical or microbiological contaminants can be used for domes-

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tic, industrial and agricultural purposes (Karl & Knight, 1998). The importance of microbiological and parasitological criteria for controlling the contamination of recycled water has been repeatedly emphasized. The most common human parasitic protozoan transmitted by water belongs to the genus Cryptosporidium (Slifko, Smith, & Rose, 2000). Cryptosporidium is an intracellular coccidian protozoan parasite that infects the gastrointestinal tract of a wide range of vertebrate hosts including humans (Xiao, Fayer, Ryan, & Upton, 2004). The disease caused by this parasite, called cryptosporidiosis, is transmitted via the faecal–oral route and can be linked to waterborne outbreaks. The clinical signs of cryptosporidiosis in humans are mainly diarrhea, dehydration, malabsorption, weight loss and/or wasting. Infection is self-limiting, but chronic infections may establish, particularly in young and immunosuppressed individuals (Fayer, Morgan, & Upton, 2000). The oocyst is the stage transmitted from an infected host to a susceptible host by the fecal–oral route. Routes of transmission can be (1) person to person through direct or indirect contact, (2) animal to animal, (3) animal to human (zoonotic infection), (4) water-borne through drinking or recreational water, (5) food-borne, and (6) possibly airborne. The United States Environmental Protection Agency´s ‘Interim Enhanced Surface Water Treatment Rule’ (IESWR) stipulates zero as the goal for the maximum contaminant level of Cryptosporidium parasite in drinking water (USEPA, 1999). In the past, stabilization ponds and lagoons have provided adequate treatment and storage at minimal costs. However, lagoons often do not provide effluents that meet regulatory standards established by federal and state authorities. In general, treatment plants utilizing sand filtration along with activated sludge have shown significantly lower levels of Cryptosporidium oocysts in finished effluents than those using activated sludge treatment alone (Madore, Rose, Gerba, Arrowood, & Sterling, 1987). The use of constructed wetlands can offer a high degree of process control allowing for the development of generally applicable design and cost criteria for a given and desired level of wastewater. Recent documentation on wetland systems for wastewater quality improvement has focused attention on the capability of constructed wetland systems to remove a wide variety of waterborne pollutants. Numerous

Water Air Soil Pollut (2007) 179:207–215

studies have shown the treatment of domestic wastewater for reduction of chemical oxygen demand (COD), biological oxygen demand (BOD), total suspended solids (TSS), phosphorous, and nitrogen (Hench et al., 2003; Kantawanichkul, Neamkam, & Shutes, 2001; Karpiscak, Gerba, Watt, Foster, & Falabi, 1996; Wiessner, Kappelmeyer, Kuschk, & Kastner, 2005). Thus, the wetland concept has become an attractive cost-effective wastewater treatment alternative compared to conventional or tertiary treatment processes. Limited information on the reduction of pathogenic microorganisms in artificial wetlands is known. Only few studies have investigated the fate of indicator bacteria and enteric viruses in wastewater applied to wetland systems (Karim, Manshadi, Karpiscak, & Gerba, 2004; Nokes, Gerba, & Karpiscak, 2003; Quinonez-Diaz, Karpiscak, Ellman, & Gerba, 2001; Reed, Crites, & Middlebrooks, 1995), therefore the present work will be conducted to address an accurate and easy procedure for detection and quantification of Cryptosporidium oocysts in domestic wastewater and to assess the removal of Cryptosporidium oocysts from domestic wastewater by using constructed wetlands.

2 Materials and Methods 2.1 Construction and operating conditions of the treatment system A pilot scale treatment system for domestic wastewater was used. It consists of the following steps in series: (1) up-flow anaerobic sludge blanket (UASB) reactor, (2) free water surface (FWS) wetland unit, and (3) sub-surface flow (SSF) wetland unit. The UASB reactor was previously described and investigated (El-Gohary & Nasr, 1999). The reactor was continuously fed with the municipal wastewater through a connection from the sewerage system of Dokki region in Cairo, Egypt. Based on results of the previous study the hydraulic retention time (HRT) was kept constant at 8 h. The feasibility of the UASB process for the treatment of raw sewage was investigated at a temperature range from 19 to 38°C. The effluent of the UASB reactor was used for continuous feeding of FWS wetland unit and consequently the effluent of FWS wetland unit was used for continuous feeding of the SSF wetland unit. The


operating conditions of wetland units are presented in (Table I). 2.2 Sample collection Samples were collected during a one-year period between January 2003 and January 2004. Samples were approximately collected every two weeks. Four sampling sites were selected (the raw wastewater and the effluent of each treatment unit). Three-liter samples were collected from each site. Samples were transported on ice to the laboratory for analyses immediately after collection. 2.3 Detection of Cryptosporidium oocysts Cryptosporidium oocysts were concentrated from wastewater samples according to the method of Shepherd and Wyn-Jones (1996). One liter from each sample was filtered through a nitrocellulose membrane (1.2 μm pore size, 142 mm diameter, Millipore Corp., USA) in a stainless steel filter holder (Schulcher-Schull, Germany). Materials which accumulated on the filter were obtained by washing the membrane with 300 ml of 0.1% Tween 80 (Sigma) in distilled water. The washing solution was collected in 50-ml plastic tubes and centrifuged at 1,500 rpm for 10 min. The combined sediments of all tubes were collected in one tube and further centrifuged at 1,500 rpm for 10 min. The supernatant was discarded and the pellet was re-suspended in 10 ml of the washing solution. Sample clarification was performed by a Percoll–sucrose gradient according to Table I Operating conditions of the wetland units Item

Free Water Surface Wetland

Subsurface Flow Wetland

HRT HLR OLR

2 days 1,380 m3 ha−1 day−1 102 kg BOD ha−1 day−1 219 kg COD ha−1 day−1 Sand (3–4 mm) 2.0 m 1.0 m 0.2 m Typha latifolia (cattail)

1 day 1,380 m3 ha−1 day−1 27 kg BOD ha−1 day−1 64 kg COD ha−1 day−1 Sand (3–4 mm) 2.0 m 1.0 m 0.2 m Typha latifolia (cattail)

Substrate Length Width Depth Plant

HRT Hydraulic retention time; HLR hydraulic loading rate; OLR surface organic loading rate.

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LeChevalier, Norton, and Lee (1991). Briefly, the 10 ml re-suspended sediments were stratified in 30 ml of Percoll-sucrose (1.15 g/l density) and then centrifuged at 1,500 rpm for 10 min. After centrifugation, the materials floated on the top of the gradient and approximately 5 ml of the Percoll–sucrose interface were drawn off immediately and diluted in 50 ml of washing solution. After further centrifugation at 1,500 rpm for 10 min, the supernatant was discarded and the pellet was spread on glass slides, air-dried, fixed with methanol 95%, and stained with acid-fast trichrome (AFT) stain according to Ignatius et al. (1997). The other two parts (1 l volume each) of each sample were separately processed in the same manner as previously mentioned in the former 1-l sample, except that the finally obtained pellet of 1 l was stained with modified Ziehl-Neelsen (MZN) stain according to Henriksen and Pohlenz (1981) and that of the last liter was preserved at −20°C to be used later on for PCR technique. 2.4 Extraction of Cryptosporidium DNA Cryptosporidium DNA was extracted according to the method of Ward, Deplazes, Regli, Rinder, and Mathis (2002) with slight modifications. In brief, the third 1 L pellet was subjected to three freeze-thaw cycles in liquid nitrogen and a hot water bath (96°C) for 1 min each. After that, proteinase K (400 μg/ml, Bio gene, USA) was added and the sample was incubated for 2 h at 56°C. After digestion, the sample was centrifuged at 4,000 rpm for 5 min to remove cell debris. Supernatant was collected and 0.6 volumes of isopropanol were added to precipitate the DNA. Samples were centrifuged at 10,000 rpm for 5 min and the supernatant was discarded. Seventy percent ethanol was added to wash the sediment followed by centrifugation at 10,000 rpm for 5 min. The supernatant was discarded and the precipitated DNA was resuspended in 20 μl of diethyl pyrocarbonate (DEPC)-treated water for PCR amplification. 2.5 Polymerase chain reaction (PCR) technique Extracted DNA and a positive control of Cryptosporidium DNA (Cryptosporidium Reference Unit, NPHS Microbiology Swansea, Singleton Hospital, Sketty, Swansea, UK) were amplified by using the PCR technique according to the method of Awad-

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El-Kariem, Warhurst, and McDonald (1994). The polymerase chain reaction mixture consisted of 5 μl 10 × PCR reaction buffer (50 mM Tris HCl, 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 50% glycerol, and 1% Triton x-100, pH 8), 6 μl (40 mM) dNTPs (Bioron, USA), 10 μl extracted DNA, 4 μl of each primer (Biosynthesis, USA): [forward AWA995 5¶TAG AGA TTG GAG GTT GTT CCT3¶ and reverse AWA1206 5¶CTC CAC CAA CTA AGA ACG GCC3¶]. The final volume of the PCR reaction was adjusted to 50 μl by addition of DEPC-treated water and overlaid with 50 μl of mineral oil. Amplification was performed in a Gene-Amp 9,600 (Perkin Elmer Corp., Foster City, CA, USA) thermocycler. Samples were subjected to an initial denaturation step of 10 min at 94°C, after which 0.5 μl (5 U/μl) of Taq DNA polymerase (Promega, USA) was added to each sample. The thermal profile consisted of 39 cycles, each of 30 s denaturation at 94°C, 40 s primer annealing at 58°C, and 40 s extension at 72°C. A final extension step of 10 min at 72°C was performed. 2.6 Analysis of PCR product PCR products were analyzed by electrophoresis in 2% (w/v) agarose gel containing ethidium bromide. In comparison with the 100 bp DNA ladder (Fermentas, Germany) the PCR product was run at 100 V for 30 min and visualized under ultraviolet (UV) light. The electrophoresis profile was determined by the Gel Doc 1000 image analysis system (Biometra, Germany). 2.7 Statistical analysis Statistical analyses were carried out using SPSSversion 7.5. Independent and paired t tests were used to make comparisons among variables.

3 Results Examination of 96 collected samples revealed that Cryptosporidium oocysts were detected in 23/24 raw wastewater samples (95.8%) by using acid fast trichrome (AFT) stain. When the same raw wastewater samples were processed and stained with modified Ziehl-Neelsen (MZN) stain a lower percentage of


positive samples 21/24 (87.5%) for Cryptosporidium oocysts was obtained (Table II). The mean count of Cryptosporidium oocysts (per 1 L) detected by the AFT stain reached 43.8, 23 and 1.3 oocysts in raw sewage, UASB and FWS effluents, respectively. Conversely, lower mean counts of Cryptosporidium oocysts in the same samples were obtained by using the MZN stain. No oocysts were detected in the SSF effluent by the two staining techniques. There was no significant difference between the two different stains in the detection of Cryptosporidium oocysts in all the treatment steps (Table III). By using PCR technique, only 11/24 raw wastewater samples (45.8%) were positive for Cryptosporidium oocysts (Table II and Figure 1), and only one sample (4.2%) was positive for Cryptosporidium oocysts in the UASB effluents (Figure 2). It contained 52 oocysts/l. Positive PCR results were not achieved in other samples containing less than 52 oocysts/l. No positive samples were detected at all in the SSF effluents by using PCR technique. The units of the pilot scale of domestic wastewater treatment system in the present work were arranged in series so as to permit primary treatment of raw wastewater through the UASB reactor. Consequently effluents of the UASB reactor was secondarily treated through FWS and SSF wetland units, respectively. By using AFT stain, the removal rate of Cryptosporidium oocysts in the UASB reactor ranged from 22.4% to 100% with a mean value of 53.1% (Figure 3). Statistically, the mean removal rate of Cryptosporidium oocysts in the UASB reactor was highly significant (P < 0.0001) by using either AFT or MZN stain (Tables IV and V). Most of Cryptosporidium oocysts which escaped from the UASB reactor and still found in the UASB reactor effluent were removed by the FWS wetland unit. The removal rate of Cryptosporidium oocysts increased in the FWS

Table II Incidence of Cryptosporidium in raw domestic wastewater by using AFT stain, MZN stain and PCR technique Samples Positive Samples Negative samples Total

Number Percent Number Percent

AFT

MZN

PCR

23 95.8 1 4.2 24

21 87.5 3 12.5 24

11 45.8 13 54.2 24


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Table III Statistical analysis of the mean count of Cryptosporidium oocysts in different treatment steps by using both AFT and MZN stain Treatment

Stain

Number of Samples

Raw

AFT MZN AFT MZN AFT MZN

24 24 24 24 24 24

UASB FWS

Mean Count/l

SD

P value

43.8 35.6 23.0 16.6 1.3 0.7

23.6 22.0 16.2 13.2 3.1 1.6

NS

NS Not significant; SD standard deviation; P probability.

Figure 1 Ethidium bromide stained 2% agarose showing Cryptosporidium PCR amplified product, the specific band length is 256 bp. a Raw wastewater samples from 1 to 12. b Raw wastewater samples from 13 to 24. M = 100 bp DNA ladder and + = positive control.

NS NS

wetland unit, it ranged from 76.3% to 100% with a mean value of 95.9% (Figure 3). Statistically, the mean removal rate of Cryptosporidium oocysts in the FWS wetland unit was highly significant (P < 0.0001) by using either AFT or MZN stain (Tables IV and V). All of the remaining Cryptosporidium oocysts which escaped from the FWS wetland unit and still found in the FWS wetland effluent were removed from the effluent by the SSF wetland unit. The mean removal rate of Cryptosporidium oocysts in the subsurface flow (SSF) wetland was 100% (Figure 3). Statistically, the mean removal rate of Cryptosporidium oocysts in the SSF wetland unit was significant by using either AFT (P = 0.05) or MZN (P < 0.05) stain (Tables IV and V).

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Figure 2 Ethidium bromide stained 2% agarose showing Cryptosporidium PCR amplified product of DNA extracted from UASB reactor effluent samples, the specific band length is 256 bp. M = 100 bp DNA ladder and + = positive control.

4 Discussion In addition to water, organic and inorganic materials, the wastewater inevitably contains pathogens excreted with faeces, urine, nasopharyngeal and pulmonary exudates. A large variety of gastrointestinal pathogens may be found in raw domestic wastewater including bacteria, viruses and parasites (Stott, Jenkins, Shabana, & May, 1997). One of the common gastrointestinal protozoa parasitizing vertebrates belongs to the genus Cryptosporidium (Slifko et al., 2000). In the present study, Cryptosporidium oocysts were detected in 95.8% of examined raw domestic wastewater samples, the results which was in accordance with that recorded by Farias, Gamba, and Pellizari (2002) who detected Cryptosporidium oocysts in all of the 24 examined raw wastewater samples collected from the Edu Chaves sewage pumping station in Brazil. Other workers in different countries recorded a lower occurrence of Cryptosporidium oocysts. Johnson, Reynolds, Gerba, Pepper, and Rose (1995) in USA detected Cryptosporidium oocysts in 54.2% of examined raw wastewater samples while Mayer and Palmer (1996) in USA detected Cryptosporidium oocysts in only 45.5% of wastewater. Both Rose, Dickson, Farrah, and Carnahan (1996) in USA and Ottoson (2001) in Sweden detected Cryptosporidium oocysts in 67% of raw wastewater samples. The occurrence of Cryptosporidium oocysts in domestic wastewater

reflects the public health condition of the surrounding population from the protozoological point of view. In the present study, the count of Cryptosporidium oocysts in raw domestic wastewater samples, stained with AFT, ranged from 0 to 83 oocysts/l with a mean value of 43.8 oocysts/l. Lemarchand and Lebaron (2003) in France found that the count of Cryptosporidium oocysts in raw domestic wastewater

Figure 3 Stepwise removal of Cryptosporidium oocysts through different wastewater treatment units by using AFT stain.


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Table IV Statistical analysis of the mean removal rates of Cryptosporidium oocysts in the different wastewater treatment steps by using AFT stain Treatment

Mean Count/l

Number of Samples

SD

P value

Pair 1

43.8 23.0 23.0 1.3 1.3 0.0

24 24 24 24 24 24

23.6 16.2 16.2 3.1 3.1 0.0

P < 0.0001

Pair 2 Pair 3

Raw UASB UASB FWS FWS SSF

P < 0.0001 P = 0.05

SD Standard deviation; P probability.

ranged from 1 to 87.1 oocysts/l with a mean value of 23.4 oocysts/l. The great diversity of the occurrence and concentration of Cryptosporidium oocysts might be due to many factors, including the prevalence and intensity of infection in the population generating the sewage and the prevailing socio-economic conditions (Carey, Lee, & Trevors, 2004). In the present study PCR technique was able to detect Cryptosporidium DNA in 11 (45.8%) out of 24 examined raw domestic wastewater samples. Positive PCR results were only achieved in samples containing 52 oocysts or more per liter. Rochelle, Ferguson et al. (1997a) and Rochelle, Leon, Stewart, and Wolfe (1997b) in USA showed that the detection limit of 1 to 10 oocysts per PCR reaction mixture was achieved only with purified preparations, but this limit reached 5 to 50 oocysts per reaction when environmental water samples seeded with Cryptosporidium oocysts were used. In Spain,Dellundé, Pina, Jofre, and Lucena (2002) found that a seeded water sample with 30 Cryptosporidium oocysts or more gave positive PCR results. In our opinion the sensitivity of PCR technique for the detection of Cryptosporidium oocysts in wastewater might be affected by the presence of undesirable organic and inorganic constituents leading to interference with the interactions between the target DNA of Cryptosporidium oocysts and DNA polymerase enzyme and consequently interfere with DNA amplification. Other workers agreed with us in that the presence of inhibitors can adversely affect PCR efficiency by interfering with interactions between DNA and DNA polymerase through various chemical and physical means (Carey et al., 2004; Tsai & Rochelle, 2001). Inhibitors of

concern in environmental samples include phenolic compounds, humic acids, and heavy metals. Constituents of bacterial cells, nontarget DNA and contaminants also may inhibit the specific amplification of nucleic acids (Carey et al., 2004). Future studies should be directed towards improving the recovery of just few oocysts from large quantities of environmental water samples, as well as improving the sensitivity of the detection methods. In the present work, the in-series connection between the two wetland units allowed complete elimination of Cryptosporidium oocysts as the first wetland unit (FWS) removed most of the oocysts present in anaerobically treated wastewater and the remaining portion of oocysts was completely removed by the second wetland (SSF) unit. Several processes may be involved in the reduction of Cryptosporidium oocysts by wetlands; sedimentation is thought to be one of these reduction mechanisms (Quinonez-Diaz et al., 2001). In a study by Karim et al. (2004) Cryptosporidium oocysts concentration was found to be 2–3 orders of magnitude greater in sediment compared with the water column, the results which suggested that sedimentation may be the primary reduction mechanism of Cryptosporidium oocysts in wetlands. Free-living filter feeding ciliated protozoa, commonly found in the constructed wetlands, may play an important role for the removal of Cryptosporidium oocysts lightly attached to sediments and surfaces. Average oocysts predation by the wetland ciliates was 10 oocysts cell−1 h−1 at high oocyst concentrations indicating that wetland ciliates may be capable of removing up to 4,670 oocysts ml−1 h−1. So, protozoan

Table V Statistical analysis of the mean removal rates of Cryptosporidium oocysts in the different wastewater treatment steps by using MZN stain Treatment

Mean Count/l

Number of Samples

SD

P value

Pair 1

43.8 23.0 23.0 1.3 1.3 0.0

24 24 24 24 24 24

22.0 13.2 13.2 1.6 1.6 0.0

P < 0.0001

Pair 2 Pair 3

Raw UASB UASB FWS FWS SSF

SD Standard deviation; P probability.

P < 0.0001 P < 0.05

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predation may be another important factor for the removal of oocysts from wastewaters in the constructed wetlands (Stott, May, Matsushita, & Warren, 2001). Brix (1997) found that macrophytes (aquatic plants) stabilize the surface of constructed wetlands, provide physical filtration through the root systems, insulate the surface against frost during winter conditions, and render huge surface areas for microbial attachment and growth. The root-substrate complexes and associated biofilm may have the capacity for filtration and adsorption of pathogens. On the other hand, shading by vegetation could reduce exposure from UV light and prevent heating of the wastewater by sunlight thus decreasing the rate of inactivation of microorganisms. Some believe that the degree to which plants contribute to the wastewater treatment is negligible in comparison to other processes within wetland systems. In conclusion constructed wetlands have been proven to be an effective low-cost technology and their uses are extremely advantageous to small communities within limited resources for wastewater treatment.

Acknowledgments We are indebted to Prof. Dr. Fatma ElGohary (Water Pollution Research Department, National Research Center, Cairo, Egypt) for her thoughtful suggestions and financial support of this work. We are also indebted to Dr. M. A. El-Khateeb (Water Pollution Research Department, National Research Center, Cairo, Egypt) for his kind assistance and great help. Deepest appreciation is due to Dr. Kristin Elwin (Clinical Scientist, Cryptosporidium Reference Unit, NPHS Microbiology Swansea, Singleton Hospital, Sketty, Swansea, UK) and Dr. Giovanni Widmer (Associate Professor, Division of Infectious Diseases, Department of Biomedical Sciences, Tufts University, USA) for their great assistance by sending us the positive control of Cryptosporidium DNA.

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