Journal of Applied Sciences Research, 6(10): 1511-1516, 2010 © 2010, INSInet Publication

Toxicity Assessment of Cyanobacteria in a Wastewater Treatment Plant, Egypt Sabah A. Badr, Mahassen M. El-Deeb Ghazy, Reda M. Moghazy Water Pollution Research Department, National Research Center, Dokki, Cairo, Egypt Abstract: Cyanobacteria are common in eutrophic natural waters. Being favored by warm, stable and nutrientenriched waters they may constitute an important part of plankton community in wastewater treatment plants (WWTP). The phytoplankton communities of two ponds (facultative and maturation) of the WWTP of El-Sadat city (Egypt) were studied with particular importance given to cyanobacteria. Mouse and Daphnia magna bioassays were performed on samples during months in which cyanobacteria dominated. During the study period (Dec. 2005March 2007) cyanobacteria were frequently dominant in that ponds ranging from 2.2- 97.8 % of total phytoplankton density, the main species was Oscillatoria spp. Toxicity bioassays were conducted during Oscillatoria dominancy period. Results revealed that Oscillatoria water-extract was toxic to Daphnia magna but not to mice. Acute 48hLC50 of daphnids was 3.15 gm/L as wet weight Oscillatoria spp. The study indicated that high counts of cyanobacteria may be source of contamination to water. Key words: Cyanobacteria, toxicity, Daphnia magna, Mice, wastewater treatment plant. INTRODUCTION The phytoplankton community is often dominated by cyanobacteria in eutrophic ecosystems. At high densities, cyanobacteria may produce toxins, endangering public health[1]. Common algae in wastewater stabilization ponds belong to Chlorophyceae, Euglenophyceae, Bacillarophyceae and Cyanophyceae [2]Cyanobacteria have been reported to dominate in some. WWTP (e.g. in Morocco: [3]and in Portugal [4]). Among them, Phormidium is a typical genus in wastewater treatments [5]. Silva et al. [2] recorded the presence of the cyanobacterium Planktothrix spp. In the Parada WWTP (Portugal), chlorophytes were dominant, although some publications suggest that cyanobacteria may produce substances toxic to bacteria [6-8], not much is known concerning which toxins are present and in what amounts. Cyanobacteria toxins may be divided into neurotoxins, hepatotoxins and dermatotoxins, according to their effects in mammals, however, the most common and most hazardous are hepatotoxins. They may cause severe liver damage and being protein phosphatase inhibitors, they appear to be potent liver tumour promoters [9]. Microcystins are now known to be produced by four planktonic cyanobacteria genera. Three are filamentous, Anabaena , Oscillatoria and now Nostoc [10] while one is coccoid; Microcystis. Microcystis produces the greatest variety of toxins differing in the

L- amino acids variant[11-12]. The cladoceran Daphnia magna is a well known standard test species in freshwater ecotoxicological studies, and detailed protocols for their use in both ecological risk assessment and prenormative tests with chemicals are available [13]. This is due to their small size, easy handling and laboratory culture, wide distribution, availability throughout the year, predominantly parthenogenetic reproduction, ecological relevance in food chains and high sensitivity [14-15]. It was established that bioassay is convenient first test for estimation of toxicity in screening of waterbloom material and laboratory cultures or cell extracts [16] . The aim of this study was to investigate the effects of water-extract of the cyanobacterial Oscillatoria sp. on the water flea; Daphnia magna and mice as test organisms. Materials and Methods 1-.Description of the study site: El-Sadat WWTP is located at 11 Km distance to the North east of the City on an area of approximately 72 feddans. The treatment plant consists of three ponds: two parallel anaerobic, facultative and three maturation (Fig.1, 2). The total hydraulic retention time for the WWTP is 28.4 days. It was designed to receive a maximal effluent flow of 33000 m3/day and surface organic loading rate 7.5-14.2

Corresponding Author: Mahassen. El Deeb Ghazy, Water Pollution Research Department, National Research Center, Dokki, Cairo, Egypt E mail [email protected]

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Fig. 1: Schematic diagram of the treatment plant at Sadat city

Fig. 2: Google earth picture of the treatment plant at Sadat city Kg/ ha/ day. This WWTP was planned for a population of 250,000 inhabitants. Almost all of the treated effluent is used for irrigation of woodlands (green belt) around the city but sometimes some effluents may remain in the storage reservoir.

3. Toxicity bioassay tests: The test organisms used in this study were two animals; the crustacean water flea, Daphnia magna and mice (mammal). The two organisms were subjected to acute toxicity testing of algal extracts.

2. Water sampling: This study started from December 2005 to June 2007 by monthly sampling from the ponds of the WWTP. Samples were collected at the surface of the ponds for identification and counting of phytoplankton and other samples were transported to the laboratory in ice box for protection from light for toxicity testing.

3.1. Daphnia magna: A freshwater D. magna strain that has been successfully grown in the laboratory in synthetic freshwater media[17], was one of the test organisms used for this study. Gravid females were transferred at regular intervals to 1-L glass beakers containing the culture medium; synthetic freshwater medium.

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The experiment was carried out in 250-ml glass beakers, containing 100 ml synthetic freshwater media for control and inoculated with 10 neonates < 24 h for both control and all treatments in this study, and placed in a 70x60x30 cm aquarium. Temperature was maintained at 22± 2º C by automatic heater (thermostat), Model "hydor", Italy. A mercury thermometer was used to measure temperature in test containers. Acute toxicity testing were conducted in triplicates, where groups of 10 < 24 h-old daphnids were placed in 250-ml beakers, each containing 100 ml medium and subjected to test conditions for 48 h. Tests were run without food addition. The number of live organisms after the elapse of 48h was recorded. Control test was run in parallel. Treatments with different concentrations of wet weight Oscillatoria (collected from the ponds and bioassay test) water-extract at 1.25, 2.5, 3.5 and 4.5 gm/L were performed to assess median lethal concentration to D. magna. AStatistical Analysis: Probit Analysis was used to calculate the 48h-LC50s for acute tests on D. magna as described by Finney[18]. The terminology recommended by Sprague[19], lethal concentration (LC) was used for survival as given here, represents an interpolation from three or more partial-effect concentrations. 3.2. Mice: The mouse bioassay is widely used as a routine analysis. It has the advantage to give a rapid answer if the algal extraction is toxic or non-toxic, which is often a requisition from the local authorities. The toxicity of the freeze-dried algal material is determined by using male mice weighing 20-23 gm. Toxicity is tested by intraperitoneal injection (i.p.) of a lysate algal cells by freeze-thawing. The material dissolved in 0.9% NaCl at a concentration of 50, 40, 30 and 20 mg wet weight per ml and suspended by vortexing for 30 min followed by 30 min centrifugation at 4000 rpm [20]. The mice were observed during the following 24 h for any signs of poisoning. RESULTS AND DISCUSSION I. Identification and quatification of phytoplankton: Cyanobacteria were more dominant in the maturation pond than in the facultative pond attaining in the maturation ponds a maximum of 97.8% of the total phytoplankton density (Fig. 3). The other important phytoplankton groups were Chlorophytes and Bacillarophytes. Cyanobacteria have been reported to be dominant in other WWTP such as Portugal [4] and

in Morocco[3] and have been used to treat wastewater in other parts of the world [21,5]. The main cyanobacteria species present in both ponds (facultative and maturation ponds) were similar and dominant by different species of Oscillatoria. This filamentous alga was dominated during February and March, 2006 and also during March, May and June, 2007 (Fig. 3). El- Sadat wastewater treatment plant may be considered as an optimal habitat for the development of cyanobacteria, and consequently as a potential source of toxins produced by these algae that may release to the environment. Mezioui et al.[7] stated that cyanobacteria in WWTP produce substances toxic to E. coli although the toxin was not quantified or identified. Other common cyanobacteria in WWTP are Synecoccus and Synchocystis [3,8]. 2-Acute toxicity test: 2-1- For Daphnia magna toxicity bioassay: Acute toxicity test (Short-Term Toxicity) was carried out by exposure Daphnia magna (neonates 24 hrs old) to water extract of the dominated blue green algae (Oscillatoria spp.) collected from the ponds and bioassay test conducted at concentrations 1.25, 2.5, 3.5 and 4.5 gm /L of wet weight of Oscillatoria sp. Ten animals were introduced to beakers contain 100 ml of each alga´s extract (triplicate beakers). A control test was applied at the same time (100 ml Daphnia magna synthetic fresh water media + 10 daphnids) effects were observed after 24 and 48 hrs by counting live D. magna neonates. Table (1) illustrated that, the extract was toxic to Daphnia. From concentration used (1.25, 2.5, 3.5, and 4.5 gm/l) 48h-LC50 was 3.15 gm/l, calculated by probit analysis [18]. 2-2-For mouse-toxicity-bioassay, male mice (20-23 gm) were used for the experiments. Five mice for Oscillatoria spp. extract were used. Mice were injected intraperitoneal (ip) with 1 ml of the test extract solution which containing equivalent final concentrations per mouse of 50,40,30 and 20 mg. Control mice were injected with 1 ml of the solution used for extraction contain only 0.9% NaCl. The injected animals were observed for different times up to 24 hrs. Behavioral symptoms and survival time were recorded by Table (2). The results showed that, the injection of Oscillatoria spp. extract into mice did not yielded any signs of neuro- as well as hepatotoxicity. Postmortem examination failed to show changes in gross morphology or appearance in any organ (Fig.4) and no any changes in the liver. (Fig. 5).

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Fig. 3: Fluctuation of the percentage of cyanobacteria in relation to total phytoplankton density in the maturation and facultative ponds of Al-Sadat WWTP. Table. 1: Effect of Blue-Green Oscillatoria sp endo-toxin on Daphnia magna concentrations gm/l Initial Neonates Number Live Neonates After ----------------------------------------------------------------24 hrs. 48 hrs. Control 10 10 10 -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------1.25 10 10 9 -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------2.5 10 7 6 -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------3.5 10 7 4 -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------4.5 10 7 3

Fig. 4: Gross Anatomy of Mice From acute toxicity test, it can be concluded that; the endocellular toxins of Oscillatoria spp. are toxic to Daphnia and not toxic to mice. The same results reported by [22] found that, a fraction of an extract from Microcystis aeruginosa, which did not contain microcystin, was much more toxic to Daphnia than another fraction containing

microcystins. In contrast, Nizan et al. [23] have demonstrated that the toxicity of Microcystis strains to mice does not always coincide with the toxicity to Daphnia. Toxins produced by cyanobacteria have been reported in marine as well as freshwater environments throughout the world.

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Fig. 5: Morphological Changes in Liver of Mice Table 2: Toxicity of Blue-Green Oscillatoria sp. Extracts on Mice Concentrations Neurotoxin (1-30 min) Hepatotoxin (45-240 min) Hepato+Neuro Toxic (15-30 min) (4-24 hrs) Control (0.9% NaCl only) NE NE NE NT --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------50 mg NE NE NE NT --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------40 mg NE NE NE NT --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------30 mg NE NE NE NT --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------20 mg NE NE NE NT NE = No Effect NT = No Toxins

Also, Pereira et al. [24] obtained negative results for cyanobacterial toxicity during the Planktothrix mougeotii bloom, using the mouse bioassay. Nizan et al. [23] studying acute toxic effects stated that various axenic strains of the cyanobacterium Microcystis aeruginosa were found to have different short term toxic effects on Daphnia magna. M. aeruginosa 7820, recorded as being toxic in the mouse assay, causes blocking of the food uptake. This mechanism does not prevent rapid mortality of D. magna, despite the small amount consumed. On the other hand, M. aeruginosa 004, also toxic to mice, did not cause mortality of D. magna, although it did block food uptake. They also concluded that the increased toxicity of aged M. aeruginosa is not due to an accumulation of extracellular metabolites, since the supernatant of these cultures showed no toxic effects. Also they assumed that it is a reflection of a qualitative or quantitative change in the intracellular content of this cyanobacterium, or both . Lampert [25], show that aged cultures of cyanobacteria are more toxic than fresh ones.

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