The most appropriate wastewater treatment is that which will produce an effluent meeting the recommended microbiological and chemical quality guidelines both at low cost and with minimal operational and maintenance requirements. Different systems are used worldwide for wastewater treatment such as activated sludge, trickling filter and waste stabilization pond systems. Pond systems are commonly employed for municipal sewage purification, especially in developing countries, due to its costeffectiveness and high potential of removing different pollutants (3), (6).
WSPs are designed to achieve different forms of treatment up to three stages in series, depending on the organic strength of the input waste and effluent quality objectives. Usually, classical WSPs consist of an anaerobic pond, followed by primary or secondary facultative ponds. If further pathogen reduction is necessary, maturation ponds will be introduced to provide tertiary treatment. WSPs are very widely used for small rural communities but large systems exist in Mediterranean basin, France and also in Spain and Portugal. However, in warmer climates (the Middle East, Africa, Asia and Latin America) ponds are commonly used for large populations (15).
In developing countries and especially in the tropical and equatorial regions like Egypt, a shortage of wastewater treatment systems is observed in rural communities. There is a great need to wastewater treatment systems to avoid the health risk problems in these communities. Wastewater treatment by WSPs has been considered an ideal way of using natural processes to improve wastewater effluents. In natural treatment systems such as WSP, the pathogens are progressively removed along the pond series with the highest removal efficiency taking place in the maturation ponds (21).
The aim of this study was to evaluate the performance of WSP in rural area in Egypt and to determine its role in the contamination of the drain.
MATERIALS AND METHODS
Wastewater treatment system in El-Mofti (Kafr El-Sheikh, Egypt) was designed to serve 3000 persons. Wastewater flow is about 225 [m.sup.3]/day mainly of domestic origin. This system consists of 500 primary septic tanks (each septic tank has approximately volume 1.8 [m.sup.3] with area 1.12 [m.sup.2] and depth 1.6 m) which used as primary treatment, a pumping station and wastewater stabilization pond which has two lines in parallel. Each line of pond consists of an anaerobic pond with volume 1400 [m.sup.3] (depth 3 m and area 475 [m.sup.2]), a facultative pond with volume 1500 [m.sup.3] (depth 1.5 m and area 1050 [m.sup.2]) and a maturation pond with volume 850 [m.sup.3] (depth 1.4 m and area 635 [m.sup.2]).
Effluents of 500 septic tanks are collected and discharged to pump station which in turn is discharged to WSP. The final effluents of WSP are discharged into El-Sabahi agricultural drain.
Sampling sites: Wastewater and water samples were collected monthly during the period from may 2005 until February 2006 at seven sites from each stages of WSP and agricultural drain which receives the final effluents of WSP. Samples from 1-4 represent: 1-influent (effluent of all septic tanks), 2-anaerobic effluents, 3-facultative effluents and 4-maturation effluents. Samples no.5-7 represent: 5-drain before mixing with treated effluents, 6-mixing point and 7-after 700 m from mixing point in El-Sabahi drain which receives the final effluent of WSP.
All samples were collected and transported within ice box and analyzed within 6 h of collection for chemical and biological examinations.
Samples analysis Physico-chemical analysis: Some physicochemical parameters such as temperature, pH, total suspended solids (TSS), chemical oxygen demand (COD), biological oxygen demand (BOD) were determined according to APHA (2) and phosphate according to Gales et al. (13). Additionally, nitrate was analyzed according to DEV (9).
Biological examination Algae: Algal growth was determined by measuring Chlorophyll a content Chl(a) spectrophotometrically and calculated according to APHA (2). Identification of algal community structure was examined by identification keys (31), (32).
Zooplankton: For zooplankton identification, few samples were filtered through a net of 55 [micro]m pore size to concentrate zooplankton in 100 ml of water but other samples containing great numbers of organisms were taken without filtration. Concentrated samples and nonfiltered samples were then preserved by Lugol's solution (20).
Zooplankton organisms were identified according to Edmondson (10) and were counted microscopically in 1.5 ml sub-samples in a Hawksley cell until attaining at least 60 individuals (23)
Bacteriological examination: Total bacterial count was determined using poured plate method while classical bacterial indicator (total coliform TC, faecal coliform FC, Escherishia coli (E.coli) and Faecal streptococci FC) were determined using MPN method. All parameters were carried out according to APHA (2) except FC and E.coli. They were carried out according to Kamel (16). Additionally, salmonellae and Listeria determination were carried out according to El-Taweel et al. (12).
Virological examination: Concentration of water and wastewater samples: All samples were concentrated by filtration through negatively charged nitrocellulose membranes according to Smith and Gerba (30) and Rose et al. (28). Then all samples were reconcentrated using an organic flocculation method according to Katzenelson et al. (18).
Nucleic acid extraction: Nucleic acids were extracted using RNA viral extraction Kit (Qiagen) according to manufacturer's instructions.
Rotavirus detection using RT-PCR: The primers VP6-3 5-GCTTTAAAACGAAGTCTTCAAC-3 and VP6-4 5-GGTAAATTACCAATTCCTCCAG-3 were used for amplification of a fragment of the VP6-coding gene corresponding to nucleotides 2-187 for rotavirus with a predicted product size of 190 bp (33).
Sequencing of amplified products: RT-PCR products of selected samples were sequenced. Fifty to one hundred [mu]l of the RT-PCR products were purified using a high pure PCR products purification kit (Qiagen) following the manufacturer's instructions. Cycle sequencing was performed on 1 to 7 ml of the purified products with an ABI prism Big dye termination cycle sequencing ready reaction kit (applied biosystem) using the same primers as in the PCR and following the manufacturer's instructions. The DNA was sequenced with an ABI prism 310 automated DNA sequencer.
Sequence data from both strands of the PCR products were aligned and compared by using the clustalw and blast programs (European bioinformatics institute).
Infection of CaCo-2 cells: Infection of CaCo-2 cells was performed as previously described (26). Briefly, after 30-min of preactivation with 10 [micro]g of trypsin/ml (grade IX; Sigma) at 37[degrees]C, samples were 10-fold diluted in PBS. Then, 100 [micro]l of direct samples or dilutions were inoculated into CaCo-2 monolayers grown in multiwell-plates (6 wells). After a 1h adsorption, a serum free overlay medium (3 ml) containing trypsin (5 [micro]g/ml) was added and the cells were placed at 37[degrees]C. Four days postinfection, cells were collected by centrifugation at 800 g, resuspended in 300 ml of PBS and freezed and thawed three times.
Detection of rotavirus infectious units using CC-RT-PCR: Rotavirus cell culture RT-PCR (CC-RT-PCR) assay was performed on suspensions of infected CaCo-2 cells. Primers VP6-3 and VP6-4 were used. RT-PCR method was the same as described previously. The detection limit in this tissue culture assay using 100 [micro]l of inoculation is 1X[10.sup.1] CC-RT-PCR units/ml, (where CC-RT-PCR units is the reciprocal end point dilution detectable by CC-RT-PCR) (1).
In this study, septic tanks were used as a pretreatment of house holds wastewater. The overall flow of wastewater to WSP which is the effluent of the septic tanks was 225 [m.sup.3]/day. The water temperature records were between 18 [degrees]C and 29[degrees]C, the average water temperature in anaerobic and facultative was 23.4[degrees]C while in maturation pond was 21.2[degrees]C. The average removal efficiencies of organic load in WSP measured as COD were 28.9, 20.24 and 9.9% after anaerobic, facultative and maturation ponds respectively. The anaerobic effluent indicated a BOD average value of 229 mg [L.sup.-1], the facultative effluents 180.7 mg [L.sup.-1] and maturation effluents 145.3 mg [L.sup.-1]. The removal efficiencies of this parameter were 22, 21.1 and 19.6% in anaerobic, facultative and maturation effluents respectively Table 1. The mean values of TSS in this system were 283.3 mg [L.sup.-1], 214.3 mg [L.sup.-1], 176.3 mg [L.sup.-1] and 157.8 mg [L.sup.-1] in influent, anaerobic, facultative, maturation effluents and the reduction of TSS was 24.4, 17.7 and 10.5% in anaerobic, facultative and maturation ponds respectively. The overall reduction of dissolved phosphorus and nitrate were 51.4 and 55.5% respectively...