Abstract: The study was carried to detect parasitic contaminants such as cysts of Giardia and entamoeba, Cryptosporidium oocysts and taenia eggs in sewage water from small lakes receiving sewage effluents from cabin toilets or directly from sewage treatment plants. There were no parasitic contaminants in the sewage water in the lake as determined by wet mount and floatation methods in Al-Ahsa. The absence of any parasite eggs in the sewage water could be attributed to the small volume of water samples taken for experimentation and to the chlorination treatment of the effluent by the sewage treatment plants. The study provided an excellent opportunity for further investigation to determine different types of water contaminants by applying other methods (specific polymerase chain reaction, PCR-RFLP methods, wound fiberglass cartridge filters and reverse transcription-PCR and commercial enzyme-Immunosorbent assay) in the sewage water in Al-Ahsa Oasis.
INTRODUCTION
Land disposal of sewage effluent and its subsequent use for different purposes including irrigation is an important environmental issue. Sewage water contains organic and inorganic contaminants. Among the various contaminants, water based parasitic diseases are caused by a number of aquatic parasites those spend part of their life cycle in the water and another part as parasites in animals and man. They include guinea worm (dracunculiasis), paragonimiasis, colonrchiasis and schistosomiasis. On the other hand, parasicitic water-borne diseases which are transmitted primarily by fecal-oral route are the major source of contamination for water through contact with human and animal fecal pollution. These include giardiasis, taeniasis, amoebiasis as well as coccidiosis. Since, these contaminants are usually present in low concentrations in contaminated water, they have to be concentrated from large volumes of water before detection. Methods of concentration include filtration through yarn-wound filters or cellulose acetate filters and retained particulates are then eluted from the filters and reconcentrated by centrifugation. The polluted parasitic stages are then separated from particulate debris by floatation (Schaefer, 1996). Subsequent detection methods, particularly for parasitic protozoa, involve immunofluorescent staining of filtered sample concentrates (Schaefer, 1996), PCR (Mahbubani et al., 1991, 1992; Webster et al., 1993) and ELISA (Da la Cruz and Sivaganesan, 1995).
Uneke and Uneke (2008) found oocysts of Cryptosporidium species in all the sampling sites and there was significant variations` in the concentration of oocysts (F-ratio = 3.367, p<0.05) with the highest mean oocyst concentration of 183.3 L-1 of water, while the least mean oocyst concentration of 120.6 L-1 of water recorded. The differences in the mean monthly oocysts concentration per litre of water was statistically significant (F-ratio = 3.23, p<0.05).
Oocysts are present in many environmental waters because Cryptosporidium is not only a human pathogen but also a zoonotic pathogen infecting livestock, as well as feral animals, in many watersheds used as sources of drinking water (Simmons III et al., 2001). Surveys of surface water, groundwater, estuaries and seawater have dispelled the assumption that Cryptosporidium oocysts are present infrequently and in geographically isolated locations and indicate that water is a major vehicle for transmission of cryptosporidiosis (Fayer, 2004; Nwachcuku and Gerba, 2004).
Numerous studies have reported the contamination of surface water by Cryptosporidium. Sources of contamination of surface waters include sewage effluent overflows, waste-water discharges, abattoir waste, direct animal faecal deposition in waterways, indirect deposition via runoff from land grazed by livestock and wildlife, manure and effluent spreading and storm water run (Gary et al., 1983; Graczyk et al., 2000; Jellison et al., 2002; Xioa et al., 2001).
Sewage effluent may contain Cryptosporidium oocysts or Giardia cysts. The sewage contamination of water was implicated in a number of outbreaks of cryptosporidiosis and giardiasis (Craun, 1979; D'Antonio et al., 1985; Davies and Ritchie, 1948). Analysis of sewage for parasites of public health significance including Cryptosporidium and Giardia were the subject of several studies throughout the world (Carrington and Gray, 1993; Parker et al., 1993; Rose et al., 1986; Smith et al., 1994; Sykora et al., 1991). Significantly more Giardia cysts were detected than Cryptosporidium oocysts in our survey of sewage influents. Enriquez et al. (1995) investigated efficiency of tertiary sand and coal filtration and chlorination on removal of both Giardia cysts and Cryptosporidium oocysts. The concentration of Giardia cysts and Cryptosporidium oocysts detected in sewage influent is affected by the numbers of contributors (i.e., number of infected humans and animals in the community served by the STW), intensity of infection and dilution by other waste discharging to the STW. Also, attempts were made to correlate diagnosed cases of giardiasis and cryptosporidiosis with numbers of cysts and oocysts in sewage influent.
Madore et al. (1987) found average numbers of oocysts detected in raw water and treated sewage effluent were 5.18nx103 and 1.30x103 L-1, respectively. Filtered sewage effluents had significantly lower number of oocysts (10.0 L-1). The results indicated that sand filtration may reduce the concentration of this parasite in waste waters. Highly variable oocyst numbers were encountered in surface waters. McHarry (1984) found Giardia sp. cysts at levels of 4,000-450,000/ 378,500 L (100,000 gallons) in sewage effluents from three of seven sewage treatment plants in sangamon county, illinois, in June, July and August 1981. Robertson et al. (2000) conducted a 3 year study on Cryptosporidium oocysts and Giardia cysts in sewage. The results demonstrated that, over 1 week, none of the environments tested had a deleterious effect on oocyst viability, with the exception of the sludge-holding tank. Similar studies were also conducted to detect oocysts of Cryptosporidium in sewage effluents (Robertson et al., 1992; 1993a, b, 1995).
Kaucner and Stinear (1998) described a Reverse Transcription-PCR (RT-PCR) for detecting low numbers of viable Cryptosporidium parvum oocysts spiked into clarified environmental water concentrates. Xiao et al. (2004) reported the presence of Cryptosporidium oocysts in 67-100% of wastewaters, 24-100% of surface waters and 17-26.8% of drinking waters. Farm animals and human sewage discharge are generally considered to be the major sources of surface water contamination with C. parvum (Da la Cruez and Sivaganesan, 1995). Because Cryptosporidium infection is common in wildlife, it is conceivable that wildlife can also be a source for Cryptosporidium oocysts in waters (Webster et al., 1993).
The information on parasitic contaminants in sewage water is inadequate for establishing criteria on the use of treated or untreated sewage effluent in Al-Ahsa Oasis, Saudi Arabia. The main objective of this investigation was to detect parasitic contaminants such as cysts of Giardia and Entamoeba, Cryptosporidium oocysts and Taenia eggs in the sewage water from Al-Ahsa Oasis, using Wet Mount and Floatation methods.
MATERIALS AND METHODS
A total of twenty water samples were collected from a small lake in Al-Ahsa Oasis receiving the sewage water from Hofuf city during 2007-2008. The site was selected because Al-Ahsa Oasis in the largest Oasis in the Arabian Peninsula having surface irrigation network. The Oasis has two main lakes (D-1 and D-2) which receive drainage effluent (agricultural drainage water, sewage water, gray water, industrial effluent etc) from the Oasis. The lakes are in an open areas and are good source of environmental pollution. Also, the drainage water after preliminary treatment is used for irrigation. The samples were randomly taken from five different locations in the lake. Four different specimens namely wet soil, mud, turbid water and clean water were collected from each sample location. Each sample was subjected to direct wet mount and centrifugation floatation techniques (McHarry, 1984; Webster et al., 1993; Xiao et al., 2004). The parasitic contaminants such as cysts of Giardia and entamoeba and Cryptosporidium oocysts were detected by following the methods of Abbaszadegan et al. (1991) and Campbell et al. (1992). Analysis of water samples was done using the techniques described in earlier studies with slight modifications (Lechevallier et al., 1991; Wohlsen et al., 2004; Njoku et al., 2005).
Filtration
The water samples were filtered immediately after collection using a white
khaki cloth (cotton) as filters (mesh size 250-300 μm) with the aid of
a vacuum pump and a Buckner filter flask. The cloth was cut into 8 cm3
pieces and to enhance an effective trapping of particles and the organisms during
the filtration process; 5 of each piece was placed together in the funnel thus
forming 5 layers. When the pores are blocked during the filtration process as
indicated by the slow flow rate of filtrate into the Buckner filter flask, the
topmost layer of the cloth was removed and another added at the bottom thus,
maintaining the five layers at any point in time as the filtration continued.
All the filters, which had trapped minute particles, were collected into marked
polythene bags and stored in the refrigerator to keep them moist.
Backwashing
The filters were eluted by several backwashing with 200 mL of distilled
water in which 10% wash solution concentration had been incorporated so as to
enhance the recovery of oocysts. The wash solution was prepared using distilled
water and Tween 20 (Sigma Chemical Co., St. Louis, Mo.).
Concentration
The sucrose gradient sedimentation technique was used for the concentration
of oocysts. After the backwashing, 9 mL of the water sample was placed in a
conical centrifuge tube and 2 mL of Sheather's sugar solution was added. This
was stirred vigorously and centrifuged at 3000 rpm for 10 min. The supernatant
was carefully removed until about 2 mL is left with the sediment in the centrifuge
tube. The sediment was transferred to the microscope slides and smears made.
Microscopic Examination
Demonstration of Cryptosporidium oocysts was by microscopic examination
of smears made after the concentration of water samples, fixed with 70% methanol
and stained using the modified Ziehl-Neelsen staining technique (Henriksen and
Pholenz, 1981).
Visual Oocyst Count
The number of oocysts was determined by scanning through each slide randomly.
This was done by moving three different parts of he slide each at a time across
the x10 objective and looking out for the pinkish stained oocyst. Oocyste encountered
were confirmed using the x40 objective, by their oval or round shape and pink-red
coloration. These were counted and their numbers recorded.
RESULTS AND DISCUSSION
No parasite eggs or cysts were detected by the Wet Mount and floatation
method in the sewage water from Al-Ahsa Oasis (Table 1).
Lakes receiving sewage effluents from cabin toilets or directly from small sewage water treatment plants are expected to harbour protozoan infective stages, such as cysts of Giardia and Entamoeba, oocysts of Cryptosporidium and Taenia eggs. Also, protozoan cysts deposited on soil from animals can contaminate such lakes during rainy season. Furthermore, aquatic helminthes, the causative agents of water based diseases, are expected to be present in such lakes. Although, the lake is formed mainly from different types of discharged effluents, yet all the sewage water samples did not show any parasitic contaminants.
| Table 1: | Parasitological examination of water samples from lake |
This could be attributed to the volume of water samples which was small and reduced the chances to detect these contaminants (Cysts and Cryptosporidium oocysts and others). The results agree with those of Schaefer III (1996) who reported that these parasites are usually present in low concentrations in contaminated water and must be concentrated from large volumes of water before detection. The study emphasized that the sensitivity of the detection techniques such as immunofluorescent staining (Schaefer III, 1996), PCR (Mahbubani et al., 1991, 1992; Webster et al., 1993) and ELISA (Da la Cruz and Sivaganesan, 1995) should be used to examine water samples for various contaminants. The results did not agree with those of Uneke and Uneke (2008) who found Oocysts of Cryptosporidium species in all the sampling sites with the highest mean oocyst concentration of 183.3 L-1 of water while the least mean oocyst concentration of 120.6 L-1 of water recorded. The absence of parasitic contaminants in sewage water of the present study are supported by the results of Enriquez et al. (1995), who investigated efficiency of tertiary sand and coal filtration and chlorination on removal of both Giardia cysts and Cryptosporidium oocysts.
CONCLUSION
There were no detectable contaminants determined by wet mount and floatation method in the sewage water of Al-Ahsa Oasis. This could be attributed to the chlorination of sewage effluent which might have removed both Giardia cysts and Cryptosporidium oocysts. Overall, the study provided an excellent opportunity for future investigations of various types of parasitic contaminants in the drainage water in Al-Ahsa Oasis by following different techniques. Because the drainage water is being used indiscriminately for irrigation and can cause health harzards among the farming community.