Abstract: Phytoplankton and microbiological analysis of three different sites of each of El-Manzala Lake Water and Rashid branch of the River Nile of Egypt were examined. The results were compared and correlated with each other and with certain physico-chemical parameters using MVSP Ver. 3.1 program for Canonical Corresponding Analysis (CCA). Chlorophyceae and Bacillariophyceae represented 47.6 and 45.9% of the total phytoplankton in El-Manzala Lake Water, which was more, polluted than Rashid estuary water. Chlorophyceae was predominant (51.7%) in Rashid estuary water. Heterotrophic plate counts and indicators of faecal contamination were higher in El-Manzala than in Rashid water whereas both halophilic and halotolerant bacteria were higher at Rashid than El-Manzala locations. Thermophilic and/or thermotolerant fungi and bacteria were more abundant in El-Manzala water than in Rashid water.
Introduction
Human population growth has significantly altered the environment for many natural water bodies. As a result the composition of the biota of these water bodies is affected (Gab-Allah, 1991 and Touliabah, 1996).
Rivers, reservoirs and estuaries are ecologically deteriorated due to unabated discharges of pollutants. The biological communities vary from one location to another on the coastal areas (Sarno et al., 1993). This is probably the result of land clearing, agriculture and urbanization as well as the alteration of the hydrological cycles (Vitosek et al., 1997). The effects of agriculture land use on streams include the run-off chemical and sediment and the alteration in channel morphology (Skinner et al., 1977).
Rashid branch is one of the Nile bifurcates which starts 23 Km north of Cairo. It runs about 236 Km northward along the west boundary of the Nile, Delta and opens in the Mediterranean Sea 12 Km north of Rashid city. The human and agricultural wastes of this city gets into the water of Rashid branch of the Nile.
El-Manzala Lake is a brackish shallow lake of 1.00B1.50 m depth, 250 Km north east of Cairo and its water is productive (Bishai and Yousef, 1977).
Rashid branch of the river Nile and El-Manzala lake are subjected to change in their environment as a result of the discharge of human and/or agricultural wastes.
Phytoplankton has been used to monitor the change in the environmental conditions in water bodies (Winter and Duthie, 1998). Thus the changes in phytoplankton communities structure along water bodies were used to assess the effect of urbanization on water quality (Winter and Duthie, 1998). Bacterioplankton, phytoplankton and zooplankton were also used to follow the change of the River Danube (Hungary) by Katalin et al. (1994).
The present investigation aimed at examining the phytoplankton, microbial indicators of faecal contamination, thermophilic and/or thermotolerant organisms and halophilic and/or halotolerant bacteria present in Rashid estuary and El-Manzala water bodies at three different sites. Certain physico-chemical parameters at the time of investigation were determined.
Materials and Methods
Samples: Water samples were collected from two-locations viz. El-Manzala Lake Water and Rashid branch water body of the River Nile in three different water sites of each location as follows:
El-Manzala lake water:
| Site I: | At El-Gamil which is directly on the border line of the Mediterranean Sea and El-Manzala Lake north of port Said City |
| Site II: | At Genka where water flows from the northern part with drainage channels (Bahr El-Baquer, Hadous, Ramsis, etcÿÿ). |
| Site III: | Close to El-Mataryia City and opposite to Rashid where agricultural wastewater as well as human wastewater are drained |
| Fig. 1: | Map showing different sites of El-Manzala (I, II, III) and Rashid locations |
| Site I: | Directly at the connection of the Mediterranean and the River Nile. |
| Site II: | An Estuary of the Mediterranean and the River Nile water (3 Km from Rashid City). |
| Site III: | Opposite Rashid City. |
The sites of each location are represented on the Map (Fig. 1).
The water samples were collected during the autumn season of 1999. The samples were always collected at 30 cm depth from the surface in sterile well stoppered bottles and transferred in an ice box to the lab. Bacteriological analyses of the collected samples were carried out within 24 hrs. Samples for the phytoplankton analysis were collected as described (American, Public Health Association, APHA, 1995) and were immediately fixed by Lugol’s Iodine.
Physico-chemical analysis: Water temperature was measured at the time of sampling using a mercury thermometer graduated to 0.1EC. The Hydrogen Ion concentration of the water of each site was measured by using a portable pH meter. Chlorosity was determined by Mohr's methods as in Vogel (1953). Salinity was calculated by multiplying the values of chlorosity by 1.80655. Dissolved oxygen was determined according to Winkler method as described APHA (1995). Total alkalinity, nutrients (nitrate, nitrite, ammonia and phosphate) and silicate were carried out as described (APHA, 1995).
Analysis of the biota: Phytoplankton biota was enumerated by the drop method (Environmental Protection Agency, EPA, 1979) and were identified according to Mizuno (1990) and Compere, (1991).
All the bacteriological analysis was carried out according to APHA (1995) for indicators of faecal pollution and heterotrophic plate count (HPC). The indicators of faecal pollution determined in the present study were; total coliform, faecal coliform, faecal Streptococcus, Pseudomonas aeruginosa and Yersinia enterocolitica.
The medium used for counting halophilic and halotolerant eubacteria was prepared according to Kamekura et al. (1985). It contained (g/100 ml) NaCl to give from 0.8 to 4.3 M; KCl, 0.02; KH2PO4, 0.01; Mg SO4.7H2O, 2.0; Na-glutamate monohydrate, 2.0; biotin 5 μg; thiamin HCl 40 μg and choline chloride 2 mg; Final pH, 7.3. While the medium used for counting archaebacteria was prepared according to Kushner, (1993), it contained (g/100 ml): NaCl to give from 0.8 to 4.3 M; KCl, 0.2; MgCl26H2O, 2.0; CaCl2.6H2O; 0.02; Yeast extract, 0.5; tryptone, 0.5; final pH 8.5.
Thermophilic and thermotolerant fungi and bacteria were counted on Sabouraud and nutrient agar, respectively. Incubation of these media continued for 24–48 hrs at 40, 45, 50, 55 and 60°C.
Statistical analysis: Canonical Component Analysis (CCA) were performed by using MVSP Ver. 3.1 Computer program between the environmental variables and the different groups of biological data. The purpose of CCA was to answer the question why these biological data aggregated in the same patterns as well as to correlate some selected physico-chemical variables with the biological features.
Results and Discussion
Physico-chemical parameters: The analytical data of the physico-chemical parameters of the examined locations indicated that the pH values of Rashid Water body were on the alkaline side (pH 8.1-8.6) whereas that of El-Manzala sites were slighty acidic (6.72-6.74) (Table 1).
| Table 1: | Some Physico-chemical parameters of the investigated localities |
*Alk = Alkalinity; S% = Salinity; DO = Dissolved Oxygen; COD = Chemical Oxygen demand; NH4 = Ammonia; NO3 = Nitrate; NO2 = Nitrite; PO4 = Phosphate; Si = Silicate. | |
| Fig. 2: | Percentsge abundance of phytoplanktonic groups at the investigated localities |
This is in agreement with the findings of Zaghloul, (1988) and Gab-Allah, (1991). The values of DO at both locations (Table 1) represented supersaturated values according to Zustshi (1976). The total alkalinity (measured as CaCO3 mg l–1) was generally higher at Rashid sites especially at site II than at El-Manzala sites (Table 1) which agreed with the results of other investigators (Bishai and Yousef, 1977; Zaghloul, 1988; Gab-Allah, 1991). According to the data of alkalinity, both locations are considered as productive water (Huet, 1973; Train, 1979).
Total salinity was also higher in certain sites of Rashid locations as compared to the corresponding sites of El-Manzala (Table 1). Gab-Allah, 1991 also reported lower salinity for El-Manzala than that for the adjacent area of the Mediterranean Sea. The salinity in site I of both locations was higher than that in sites II and III of the same locations (Table 1). This is mainly due to the location of site I which is very close to the Mediterranean (Fig. 1) and the dilution of sites II and III with fresh water of the River Nile and/or the water from the drainage systems.
The chemical oxygen demand (COD) was always higher for El-Manzala location as compared to Rashid Water and for site III in each location as compared to site II and site I (Table 1). The high COD values for El-Manzala is a direct result of the pollution of this location (especially sites II and III) by the drainage system of Bahr El-Baquer, Hadous and Ramsis. Phosphate concentrations were higher for El-Manzala location than for Rashid and for site I than site II and III for El-Manzala while for Rashid Water it was high at site II. On the other hand, each of ammonia, nitrite and nitrate concentrations were higher in El-Manzala comparable to Rashid sites. The concentrations of these ions decreased gradually from site III at each of the above two locations to sites II and I. This is another indication of the high pollution in site III for both locations due to the drainage and/or human activities (Bishai and Yousef, 1977; Touliabah, 1996).
Phytoplankton and microbiological analysis: Data showed that the identified algae belong to six main groups namely; Bacillariophyceae, Chlorophyceae, Cyanophyceae, Dinophyceae, Euglenophyceae and Cryptophyceae.
| Fig. 3a-f: | Heterotrophic plate count (HPC) and the indicators of faecal pollution |
The largest phytoplanktonic group in El-Manzala Lake Water fluctuated between Chlorophyceae and Bacillariophyceae (47.61 and 45.95% of the total phytoplankton, respectively, Fig. 2). On the other hand, in Rashid estuary water, the most dominant phytoplanktonic group was Chlorophyceae, which constituted 51.71% of the total phytoplankton followed by Bacillariophyceae (36.57%). In both selected locations, it was found that Euglenophyceae and Cryptophyceae were rare in different sites and represented 0.49 and 0.17% respectively of the total phytoplankton at El-Manzala sites and 0.33 and 0.40% respectively at Rashid estuary water.
The general pattern of phytoplankton standing crop in El-Manzala Lake water was considerably high (2.75H107 cells l–1) if compared with the corresponding values of Rashid estuary (9.08H106 cells l–1). Results also indicated that, in case of El-Manzala Lake water the maximum total phytoplanktonic standing crop was recorded in site III which formed 47.73% of the total phytoplankton crop, while the minimum crop was observed in site I (1.66%).
| Fig. 4: | Distribution of halophilic bacteria at the investigated localities |
On the other hand, the total phytoplankton standing crop at Rashid estuary fluctuated from 3.91×106 cells l–1 in site III to 1.91×106 cells l–1 in site I. The standing crop of phytoplankton of the marine sites were less than those recorded in both sites II and III in the studied locations. The results of the present investigation agreed with the findings of Gab-Allah, (1991). She stated that, Lake El-Manzala could be classified as a hyposaline and most organisms inhabiting the lake were typically freshwater and originate from the River Nile except at the northern part at El-Gamil. It connects the lake with the open sea and may contain some marine forms. On the other hand, Rashid estuary (site I) contained less phytoplankton standing crop than those recorded in sites II and III. This may be a direct effect of influxes of drainage water particularly in site II and /or site III. This view agrees with the results of Zaghloul (1988).
The most leading species of Chlorophyceae in both locations were Chlamydomonas gracilis, Actinastrum hanzschii, Ankistrodesmus falcatus, Scenedesmus quadricauda and Tetraedron minimum. While the most dominant species of Bacillariophyceae were; Cyclotella meneghiniana, Nitzschia closterium, N. palea and Navicula sp. Most of these species were recorded by (Zaghloul, 1988 and Gab-Allah, 1991).
Cyanophyceae were recorded in both locations and represented as a number of units. Each unit equals 100 μm (EPA, 1979). The standing crop of Cyanophyceae in El-Manzala Lake was higher than that recorded in Rashid estuary (2.70 and 1.47% of the total phytoplankton respectively). The most leading species of this group were Merismopedia tenuissima, Microcystis aueriginosa, M. flos-aquae, Lyngbya limntica and Oscillatoria limnetica.
| Fig. 5: | Distribution of thermophillic micrograinsims at the investegated lovalities |
| Fig. 6a: | Phytoplankton groups and environmental data |
| Fig. 6b: | Heterotrotrophic plate count and indicators of faecal contamination variable |
| Fig. 6c: | Heterotrophic plate count and indicators of faecal contamination variables |
The same phenomenon was recorded with Dinophyceae, which represented 3.13 and 2.92% of the total phytoplankton respectively in El-Manzala Water and Rashid estuary. The most dominant species of this group were Exiuvilla apora, Peridinium cinctum, Gymnodinium fusum and Ceratium hirdinulla.
Generally speaking the heterotrophic plate counts (HPC) in El-Manzala sites were higher than those in Rashid sites. The highest counts in both locations were recorded in site III, this was followed by site II and site I (Fig. 3). The same observation was true for the total coliform, the faecal coliform, the Streptococcus and the Pseudomonas aeruginosa counts. However, Strep. faecalis, total and faecal coliform were absent in site I for El-Manzala location, while Pseudomonas aeruginosa was not detected in site I in both locations as well as site II and site III for Rashid locations (Fig. 3). This might suggest a greater sensitivity of these organisms to high salt concentrations. The high total HPC as well as the faecal indicator organisms are a direct indication of pollution. Site III in both locations is probably highly polluted with human and agricultural wastes (from Bahr El-Baqer, Ramsis and Hadous drainage canals in El-Manzala location and from Rashid city in Rashid locations). This is also suggested by the relatively high concentrations of ammonia, nitrite, nitrate and silicate in site III in both locations as compared to site II and I (Table 1). Site III was also very low in salinity in both locations as compared with sites II and I. It should also be mentioned that total coliform as well as faecal coliform, faecal sterptococci and Pseudomonas aeruginosa completely disappeared from site I in El-Manzala location which might suggest that this site is free of faecal pollution (Fig. 3). On the other hand, faecal coliform as well as faecal streptococci were present at site II and III for both locations and were usually higher in El-Manzala locations than Rashid ones (Fig. 3), suggesting a higher pollution for the former than the latter locations. This also suggests a synergestic relationship between Strep. faecalis and faecal coliform. This has been also suggested by Gale, (1943).
Kushner (1993), indicated that the medium suitable for the growth of halophilic eubacteria is different from that for the growth of halophilic archaebacteria. In the present investigation both media were used, the results (Fig. 4) indicated that among the halophilic and/or halotolerant bacteria, archaebacteria were more abundant than eubacteria. The frequency of the presence of halophilic and/or halotolerant bacteria in general was more for the Rashid location than for El-Manzala one (Fig. 4). The pH of Rashid location was higher than that for El-Manzala one (Table 1). Oren (1993) reported that archaebacteria prefers pH range (8.0-9.5) for its growth. Thus halophilic and/or halotolerant eubacteria were not detectable in El-Manzala in all sites and counts of archaebacteria were usually higher for Rashid than El-Manzala in corresponding sites. Counts of archaebacteria capable of growth at 2.5 M NaCl were low in site I and disappeared completely in El-Manzala (sites II and III). The concentrations of silicate, nitrite and nitrate decreased significantly in site III to site II and was lowest in site I. This was more obvious in El-Manzala than in Rashid locations. Counts of halophilic and/or halotolerant archaebacteria showed exactly the reverse trend. These counts were higher in site I in both locations. It decreased gradually to the lowest value in site III. The amounts of nitrate and silicate were usually higher in El-Manzala than in Rashid in the corresponding sites. The possible inhibitory effects of ammonia, nitrite and nitrate concentrations on halophilic and/or halotolerant bacteria needs to be investigated.
The results of statistical analysis (Fig. 6a-c) indicated that archaebacteria capable of growth at 5% NaCl are directly proportional to archaebacteria capable of growth at 10 and 15% NaCl. All these groups of archaebacteria is proportional to Cryptophyceae and Dinophyceae. It is known that both Cryptophyceae and Dinophyceae are stimulated at higher salt concentrations. Rashid sites were higher in their salt concentrations than El-Manzala sites (Table 1). Generally, counts of halophiles and halotolerant bacteria were more frequently encountered in Rashid sites than El-Manzala (Fig. 4). On the other hand, eubacteria capable of growth at 5% salt and 10% salt are proportional to cyanobacteria (blue-green algae). The latter group is stimulated in the presence of high concentration of organic matter. At the same time the halophilic and halotolerant eubacteria requires certain organic compound (such as biotin) in their culture medium.
Fig. 5 indicated that thermotolerant and/or thermophilic bacteria are more abundant than thermophilic fungi. It also indicated that the counts of these groups were generally higher in El-Manzala than in Rashid sites at relatively higher temperature (55-60°C). These organisms were not detected in Rashid samples in all locations. Bacteria and fungi capable of growth at 55°C were only encountered in samples of site III in El-Manzala. It was also interesting to notice that bacteria capable of growth at 60°C were only isolated from site I of El-Manzala. Site I contained the lowest amount of nitrite, nitrate and ammonia as compared to the other two sites. This might have an effect on the occurrence of bacteria capable of growth at 60°C. Further investigation must be carried out to clarify this observation.
Bacteria and fungi capable of growth at temperature higher than 45°C were not detected in samples of sites II and III in Rashid locations while sites II and III of El-Manzala supported bacteria and fungi capable of growth at 55EC. Fungi capable of growth at 60°C were not detected at any of the examined sites. Generally speaking, thermophilic fungi capable of growth at 60°C or higher are not common (Suberkropp, 1984; Sridhar and Barlocher, 1993).
Analysis of biota
Statistical analysis: Fig. 6 (a, b and c) shows the graphical representation of Canonical Corresponding Analysis (CCA) for axis 1 and 2. Community structure and distribution of phytoplankton classes along the environmental variables are clear (Fig. 6a). The diagram indicates that salinity, orthophosphate are more effective and positively correlated with Cryptophyceae, Dinophyceae and Bacillariophyceae (Diatoms). Cyanophyceae (Blue-green) correlated and was effected by Chemical Oxygen Demand (COD). While nitrite, ammonia, nitrate and total alkalinity affected and correlated with Euglenophyceae. This finding supported the conclusion of El-Naggar et al. (1997). They stated that Euglenophyceae were directly affected by organic matter.
In general, total phytoplankton and Chlorophyceae are considered as floating biotic variables, which was affected by most of the environmental variables (physico-chemical parameters). For CCA diagram, the direction and length of each arm play an important role within the biotic variables that occurred at the same pattern and inversely related with those at the other pattern.
The (CCA) of the indicator of faecal pollution were affected by most environmental parameters. Other investigators (LeChevallier et al. 1980; LeChevallier and McFeters, 1984; McFeters et al., 1982) concluded that the number of chemical and physical factors common to water systems are known to cause a form of sublethal and reversible injury that is responsible for the failure of water borne coliforms to grow on accepted media.
The graphical representation of (CCA) showing the distribution of various group of the biota and their relation to each other (Fig. 6-c) indicated that the following groups of microorganisms are positively correlated with each other: Archaebacteria at 5, 10 and 15% salt concentrations; Dinophyceae and Cryptophyceae. It is known that most of these organisms tolerate high salt concentration. The results of the present study are in agreement with the findings of Carole and Waaland (1988). They stated that, some Dinophyceae could be maintained in restricted conditions like high salinity and the maintenance of salinity gradient by freshwater to salinity water. Further investigation is needed however, to study the effect of high salt concentrations on fungi capable of growth at 50°C which is also correlated with these groups (Fig. 6-c).
Furthermore, fungi at 40°C, bacteria at 45°C and 50°C and eubacteria at 10% salt concentration and Cyanophyceae are all positively correlated with each other (Fig. 6c). At present there is no direct explanation for the presence of these groups together. It should be mentioned, however, that Cyanophyceae is usually as indicator of pollution with organic matter and halophytic or halotolerant eubacteria require organic compounds in their media of growth.
The associative growth of the above mentioned microbial flora and their metabolic influences on each other needs further investigation. This should help to elucidate their effect on each other and subsequently on the environment. It might provide an explanation to their presence all together in the same ecosystem under the same conditions of physico-chemical parameters in the same location. This might have some indications from the environmental point of view.