Occurrence of cyanobacteria and microcystins in waste stabilization ponds in northeast of Brazil

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1 11th IWA Specialist Group Conference on Wastewater Pond Technology, University of Leeds, March 2016 Occurrence of cyanobacteria and microcystins in waste stabilization ponds in northeast of Brazil Araújo, F. 1 ; Lima, W. R. de 2 ; Becker, V. 1 ; Araújo, A. L. C. 2, 3 ; Camargo-Valero M. A. 4, 5 1 Laboratório de Recursos Hídricos e Saneamento Ambiental, Departamento de Engenharia Civil, Centro de Tecnologia. Universidade Federal do Rio Grande do Norte, , Natal, RN, Brazil. 2 Programa de Pós-Graduação em Engenharia Sanitária, Departamento de Engenharia Civil, Centro de Tecnologia. Universidade Federal do Rio Grande do Norte, , Natal, RN, Brazil. 3 Department of Natural Resources, Federal Institute for Education, Science and Technology of Rio Grande do Norte. Natal-RN, Brazil. 4 School of Civil Engineering, University of Leeds, Leeds LS2 9JT, UK. 5 Departamento de Ingeniería Química, Universidad Nacional de Colombia, Sede Manizales. *Corresponding author, acalado@ifrn.edu.br ABSTRACT Cyanobacteria and their toxins can commonly occur in waste stabilization ponds (WSP). This study evaluated phytoplankton communities in facultative and maturation ponds of seven WSP systems in northeast of Brazil, with particular interest given to the presence of cyanobacteria and microcystin. During this study, cyanobacteria were dominant in most of tested samples, but microcystin concentrations were always bellow 0.2 g.l -1. The WSP evaluated had a low efficiency in the removal of organic matter and nutrients. Although concentrations of cyanobacteria decrease from facultative to maturation ponds, final effluents may be a source of contamination to water bodies with cyanobacteria and their toxins. KEYWORDS Cyanobacteria; microcystin; waste stabilization ponds; toxins. INTRODUCTION Waste stabilization ponds series (WSPS) are natural systems where raw wastewater is treated by a mutualistic process involving both algae and bacteria. Although used worldwide WSP are particularly adequate for tropical countries due to their favorable climate: high solar radiation and temperature (Peña and Mara, 2004). In addition, WSPS are simple to construct, to operate and maintain, and are low-cost. There are three main types of WSPS (anaerobic, facultative and maturation ponds), which are arranged in series. Series of ponds have high efficiency on organic matter and fecal bacteria removals. In addition, this system can remove some nutrients (nitrogen and phosphorus) and suspended solids. The presence of algae in facultative and maturation ponds is very important in this process due to maintenance of aerobic conditions for degradation of organic matter and the uptake of nutrients. Cyanobacteria and their toxins can commonly occur in WSPS (Furtado et al., 2009; Kotut et al., 2010; Vasconcelos and Pereira, 2001). The high availability of nutrients associated with the high temperature can favor the development of algae and cyanobacteria blooms (Paerl and Huisman, 2008). Cyanobacteria blooms represents a health problem since they are potentially toxins producers (Paerl and Otten, 2013). Once in WSPS, cyanobacteria and their toxins can be discharged directly on the receiving water bodies and few studies have addressed this

2 2 Occurrence of cyanobacteria and microcystins in WSPS issue. Here, we evaluated phytoplankton communities in facultative and maturation ponds of seven WSPS in northeast of Brazil, with particular interest given to the presence of cyanobacteria and microcystin. In addition, we evaluated the efficiency of the ponds in remove organic matter, suspended solids and nutrients. METHODS Samples were collected from seven waste stabilization ponds systems located in Rio Grande do Norte State, northeast of Brazil. The WSPS consist of a single series of a primary facultative pond (PFP), followed by two maturation ponds (MP1 and MP2). Ponds are operated by the State Water and Wastewater Company and by municipality Autonomous Water and Wastewater System (Table 1). Table 1. Morphometric and operation parameters of the Wastewater Stabilization Ponds. Characteristics Depth (m) Area (m 2 ) Volume (m 3 ) Hydraulic retention time (days) Flow rate Waste stabilization pond series - WSPS PN PV PIP CRV SA MIS SG PFP M M PFP 52,510 6,000 4,600 1,119 8,418 22,950 3,024 M1 28,028 2, ,287 7,200 1,386 M2 28,548 2, ,090 7,200 1,386 PFP 105,020 12,600 9,200 1,679 16,836 45,900 1,679 M1 42,042 3,840 1, ,431 10, M2 42,822 3,840 1, ,135 10, PFP M M , , (m 3 /day) WSPS: Ponta Negra (PN), Pedro Velho Roça (PV), Pipa (PIP), Caiçara (CRV), Santo Antônio (SA), Ilha de Santana (MIS) and Coqueiros (SG). Ponds were sampled six times between November/2012 and August/2013. Samples were collected directly from raw wastewater (RW), primary facultative pond (PFP) and second maturation pond (MP2), stored in polyethylene bottles and transported to the laboratory on icebox. Subsamples were taken for cyanobacteria identification and quantification, microcystin analysis and determination of organic matter, suspended solids and nutrient concentration. Algae samples were fixed with acetic solution of lugol. Toxins samples were stored at 4 C before analysis. Phytoplankton composition was determined by counting using an inverted microscope at 400x magnification according to Utermöhl (1958). At least 100 individuals of the most frequent species were counted (Lund et al., 1958) through random fields (Uehlinger, 1964),

3 Araújo et al. 3 with a minimum of 80% efficiency (Pappas and Stoermer, 1996), to estimate algal density (ind.ml -1 ). Cyanobacteria was defined as dominant when their contribution was above 50% of total algal biomass. Microcystin samples were freeze/thaw three times for lysing cells to release intracellular toxins, and then were filtered on 0.45 µm glass fiber filters to remove suspended solids. The samples were sonicated for 1 minute and frozen before analysis. Microcystin concentration in water were measured by ELISA (Envirologix TM Quantiplate TM kit). The organic matter was estimated by determining the concentration of chemical oxygen demand (COD) and biochemical oxygen demand (BOD). Turbidity was measured with a turbidimeter AP2000. Samples were filtered on 1.2 µm glass fiber filters for the determination of total suspended solids (TSS) concentration by gravimetric method. Total phosphorus (TP) concentration was measured with a spectrophotometer by the stannous chloride method after persulphate digestion. Ammonia-nitrogen concentration was measured with a spectrophotometer by the micro-kjeldahll method. All determinations followed the standard recommendations by APHA et al. (2005). RESULTS AND DISCUSSION During this study, cyanobacteria showed densities above 250,000 cel.ml -1 in facultative and maturation ponds for most of WSPS (Figure 1a-b) and were dominant in most of WSPS, representing more than 60% of total algae composition, except for PV series (Figure 1c-d). Chlorophyceae were the second taxon abundant in WSPS with densities below 250,000 cel.ml -1 in all samples (Figure 1a-b), counting for 0.2 to 36.9% of total algae composition (Figure 1c-d). The dominant species of cyanobacteria found were Synechocystis sp, Merismopedia tenuissima, Planktothrix sp and Microcystis sp (Table 2). Microcystin were detected in most of ponds but the concentrations were always bellow 0.2 g.l -1 (Table 3). Figure 1. Density (cel ml -1 ) and relative density (%) of phytoplankton (Cyanobacteria, Chlorophyceae and others) in WSPS: Ponta Negra (PN), Pedro Velho (PV), Pipa (PIP), Caiçara (CRV), Santo Antônio (SA), Macau (MIS) and Coqueiros (SG).

4 4 Occurrence of cyanobacteria and microcystins in WSPS Table 2. Phytoplankton dominant species in WSPS: Ponta Negra (PN), Pedro Velho (PV), Pipa (PIP), Caiçara (CRV), Santo Antônio (SA), Macau (MIS) and Coqueiros (SG). Waste stabilization pond series - WSPS Cyanobacteria PN PV PIP CRV SA MIS SG Planktothrix sp. X Synechocystis sp. X X X X X Merismopedia tenuissima X X X X Mycrocystis sp. X Table 3. Minimum and maximum concentration of microcystin in WSPS: Ponta Negra (PN), Pedro Velho (PV), Pipa (PIP), Caiçara (CRV), Santo Antônio (SA), Macau (MIS) and Coqueiros (SG). ND = no detectable. Waste stabilization pond series - WSPS Microcystin (µg.l -1 ) PN PV PIP CRV SA MIS SG Minimum ND ND ND ND ND ND ND Maximum ND The removal of cyanobacteria was low in most of ponds ( %) and in some WSPS we observed an increasing in cyanobacteria density in maturation ponds (Figure 2). The COD concentration had a tendency to decrease throughout the series, while BOD concentration reduced from raw sewage to facultative ponds and maintained the mean concentration in final maturation ponds (Figure 3a-b). Turbidity and TSS concentration had a tendency to increase from raw sewage to facultative ponds and decreasing in maturation ponds (Figure 3c-d). Total phosphorus concentration did not show a general tendency, while N-NH4 concentration reduced from raw water to facultative ponds and maintained the mean concentration in maturation ponds (Figure 3e-f). Figure 2. Removal efficiency for cyanobacteria in WSPS: Ponta Negra (PN), Pedro Velho Roça (PV), Pipa (PIP), Caiçara (CRV), Santo Antônio (SA), Ilha de Santana (MIS) and Coqueiros (SG).

5 Araújo et al. 5 Figure 3. Mean values (±SD) of the variables chemical oxygen demand (COD), biochemical oxygen demand (BOD), total suspended solids (TSS), turbidity, total phosphorus (TP) and ammonia-nitrogen (N-NH4) for raw water (RW), facultative pond (PFP) and final maturation pond (MP2) in WSPS: Ponta Negra (PN), Pedro Velho Roça (PV), Pipa (PIP), Caiçara (CRV), Santo Antônio (SA), Ilha de Santana (MIS) and Coqueiros (SG). In general, we observed a good removal efficiency for COD, BOD and N-NH4 concentration in facultative and maturation ponds for the WSPS (Figure 4a-b;e-f). However, for TSS and turbidity removals were negative for most WSPS in both facultative and maturation ponds (Fig. 4 c-d), probably due to the high algae content on ponds.

6 6 Occurrence of cyanobacteria and microcystins in WSPS Figure 4. Removal efficiency for chemical oxygen demand (COD), biochemical oxygen demand (BOD), total suspended solids (TSS), turbidity (TURB), total phosphorus (TP) and ammonia-nitrogen (N-NH4) in WSPS: Ponta Negra (PN), Pedro Velho Roça (PV), Pipa (PIP), Caiçara (CRV), Santo Antônio (SA), Ilha de Santana (MIS) and Coqueiros (SG). FINAL CONSIDERATIONS Although concentrations of cyanobacteria decrease from facultative to maturation ponds, final effluents may be a source of contamination to water bodies with cyanobacteria and their toxins. Cyanobacteria was dominant in most of WSP in both facultative and maturation ponds and microcystins were always below 2 µg L -1. Final effluents may be a source of contamination to water bodies with cyanobacteria and their toxins. In addition, an effluent rich in nutrients can cause an enrichment of receiving water bodies, leading to eutrophication and to the consequent cyanobacteria blooms. WSPs are artificial eutrophic ponds and cyanobacteria can constitute a substantial part of phytoplankton community in these systems (Aquino et al., 2010; Furtado et al., 2009; Vasconcelos and Pereira, 2001). The warm temperature in tropical climate and the high nutrient levels are suitable for algal blooms, mainly favoring cyanobacteria (Paerl and

7 Araújo et al. 7 Huisman, 2008). The dominant cyanobacteria genera found in this study (Synechocystis, Merismopedia, Planktothrix and Microcystis) were observed in others WSPS (Aquino et al., 2010; Furtado et al., 2009; Kotut et al., 2010; Vasconcelos and Pereira, 2001), and associated with the presence of microcystin in treated effluent. Facultative ponds are designed for BOD removal by the oxygen produced by algal photosynthesis and it is expected to have high efficiency (Mara, 2003). The mean removal efficiency ranged from 51-88% in this study for facultative ponds and similar result was observed for maturation ponds (57-78%). As a result of the low removal efficiency, the BOD concentration in final effluent, which had a large variation, was mostly higher than the required for treated effluent quality for discharge into surface waters (WHO, 1997). The removal efficiency of TSS was also lower than the expected for WSPS system. In this study, TSS increased in facultative ponds, and had a low removal efficiency for some WSPS in maturation ponds. The high BOD and TSS concentration in final effluent can be explained by to the high algae concentration. Phosphorus removal was low and its concentration in final effluent was usually > 3 mg L -1, which is expected for WSPS (Mara, 2003). However, phosphorus and nitrogen can lead to enrichment and to cyanobacterial blooms of surface water. This shows the need for additional treatment before discharge the effluent in receiving water bodies. In new WSPS the Water Company is considering the use of dissolved air flotation systems for the post treatment of pond effluents. The high concentration of cyanobacteria and the presence of microcystin in final effluents besides the high nutrient concentration may be a source of contamination to water bodies. This show the need for monitoring the presence of cyanobacteria in the receiving waters. REFERENCES APHA; AWWA; WEF. (2005). Standard methods for the examination of water and wastewater. Washington, D.C. 21 st Edition. Aquino, E.P. De, Lacerda, S.R., Freitas, A.I.G. De, Cianobactérias das lagoas de tratamento de esgoto no semi-árido nordestino (Ceará, Brasil) Cyanobacteria in sewage treatment ponds in semi-arid northeast (Ceará, Brazil). Rev. Botânica - J. Bot. 39, Furtado, A.L.F.F., Calijuri, M.D.C., Lorenzi, A.S., Honda, R.Y., Genuário, D.B., Fiore, M.F., Morphological and molecular characterization of cyanobacteria from a Brazilian facultative wastewater stabilization pond and evaluation of microcystin production. Hydrobiologia 627, Kotut, K., Ballot, A., Wiegand, C., Krienitz, L., Toxic cyanobacteria at Nakuru sewage oxidation ponds - A potential threat to wildlife. Limnologica 40, Mara, D., Domestic Wastewater Treatment in Developing Countries, Routledge. Paerl, H.W., Huisman, J., Blooms like it hot. Science (80-. ). 320, Paerl, H.W., Otten, T.G., Harmful Cyanobacterial Blooms: Causes, Consequences, and Controls. Microb. Ecol. 65, Peña, M.V., Mara, D., Waste Stabilisation Ponds. IRC Int. Water Sanit. Centre. Netherlands. 37. Vasconcelos, V.M., Pereira, E., Cyanobacteria diversity and toxicity in a Wastewater Treatment Plant (Portugal). Water Res. 35, WHO, Water Pollution Control - A Guide to the Use of Water Quality Management Principles, Water Pollution Control - A Guide to the Use of Water Quality Management Principles. London: E & FN Spon.