Concentrations and inactivation of Ascaris eggs and Water Science and Technology pathogen indicator organisms in wastewater stabilization pond sludge K.L. Nelson Department of Civil and Environmental Engineering, 535 Davis Hall, University of California, Berkeley, CA, 94720-1710, USA (E-mail: [email protected]) Vol 48 No 2 pp 89–95 Abstract During treatment in wastewater stabilization ponds (WSPs) many pathogens, in particular helminth eggs, are concentrated in the sludge layer. Because periodic removal of the sludge is often required, information is needed on the concentrations and inactivation of pathogens in the sludge layer to evaluate the public health risk they pose upon removal of the sludge. In this paper, previous reports on the sludge concentrations of various pathogen indicator organisms and helminth eggs are reviewed and results from our own recent experiments are reported. The advantages and disadvantages of several methods for studying inactivation in the sludge layer are discussed, as well as implications for the management of WSP © IWA Publishing 2003 sludge. In our recent experiments, which were conducted at three WSPs in central Mexico, sludge cores, dialysis chambers, and batch experiments were used to measure the inactivation rates of fecal coliform bacteria, fecal enterococci, F+ coliphage, somatic coliphage, and Ascaris eggs. The first-order inactivation rate constants were found to be approximately 0.1, 0.1, 0.01, 0.001, and 0.001 d–1, respectively. The concentrations of all the organisms were found to vary both vertically and horizontally in the sludge layer; therefore, to determine the maximum and average concentration of organisms in the sludge layer of a WSP, complete sludge cores must be collected from representative locations throughout the pond. Keywords Ascaris eggs; pathogen inactivation; sludge; wastewater stabilization pond Introduction Wastewater stabilization ponds (WSPs) are widely used throughout the world because they are a simple, low-cost, low-maintenance process for treating wastewater. Due to the sedimentation of suspended solids, sludge accumulates in the bottom of ponds such that periodic sludge removal is usually required, particularly from primary ponds. Although stabilization occurs during the time that the sludge is stored in the pond, the sludge may contain significant concentrations of pathogens. The concentrations of these pathogens must be known to estimate the risk they pose upon removal of the sludge. Furthermore, information on the distribution of pathogens and their inactivation rates is needed for evaluating sludge management strategies. The principal pathogens of concern in WSP sludge are helminth eggs, which are concen- trated in the sludge layer due to their high settling velocities. One species, Ascaris, has an extremely high prevalence in most developing countries; it has been estimated that over 1 billion people are infected worldwide (Crompton, 1999). Protozoan cysts, as well as bacterial and viral pathogens attached to particles may also accumulate in the sludge layer via sedimentation. Rather than measure these pathogens directly, indicator organisms are typically used. Fecal coliform bacteria and Enterococci are common indicators of enteric bacterial pathogens, whereas F+ coliphage has been used as an indicator of enteric virus (Havelaar et al., 1993; IAWPRC and Study Group on Health Related Water Microbiology, 1991; Turner and Lewis, 1995). In this paper, previous reports on the WSP sludge concentrations of various pathogen indicator organisms and helminth eggs are reviewed followed by results from our own 89 recent experiments. The advantages and disadvantages of several methods for studying inactivation in the sludge layer are discussed, as well as implications for the management of WSP sludge. Literature review Wastewater stabilization ponds can achieve nearly complete removal of helminth eggs if K.L. Nelson properly designed and operated. Ayres et al. (1992) developed an empirical design equation for the removal of human intestinal nematode eggs1 from WSPs using data from several ponds in Kenya, Brazil, and India. In addition to the removal efficiency, the most important factor affecting the concentration (and types) of helminth eggs in the sludge layer is prevalence in the community (Lloyd and Frederick, 2000). Because the prevalence of helminth infections varies widely between communities (O’Lorcain and Holland, 2000), and because the removal efficiency of helminth eggs in ponds varies, their concentrations in the sludge layer are expected to vary widely from pond to pond. Therefore, regional and even pond-specific data on the concentrations of helminth eggs in WSP sludge are needed. The few existing published data on the concentrations of helminth eggs and indicator organisms in WSP sludge illustrate the wide range of concentrations that may be found. The concentration and distribution of helminth eggs were measured in an experimental, facultative, wastewater stabilization pond in NE Brazil after 2.5 years of operation (Ayres et al., 1993; Stott et al., 1994). Sludge cores were collected on a 3 × 5 grid after draining the pond, and the average concentration and egg viability were determined in each core. The concentration of eggs was extremely high, ranging from 5,000 to 44,000 eggs/g dry weight (of which 90% were Ascaris sp.), due to the high concentration of eggs in the raw wastewater, averaging around 38,000 eggs/L. The maximum concentration of eggs was found at a distance about one-third of the length of the pond from the inlet; this distribution is consistent with the predicted behavior of an ideal sedimentation basin. Although Ayres et al. did not measure the inactivation rate of the eggs, they did compare the percentage of viable eggs in the top 5–10 cm of sludge (53%) with the average value in one entire sludge core (7.3%), demonstrating that a significant degree of inactivation had occurred in the deeper sludge. Data on helminth eggs in sludge from three WSPs in SE Brazil have also been reported (Franci, 1999). The average concentrations were 76, 13, and 13 eggs/g TS, much lower than in the pond in NE Brazil. The percentage of viable eggs was reported to be about 5% in the first pond, 23% in the second, and was not determined in the third. In the first pond, the dis- tribution of eggs was studied by collecting samples from eight different locations through- out the pond; the highest concentration was near the outlet (~ 300 eggs/g TS). Because this pond was anaerobic, it is likely that the detention time was shorter than that in the faculta- tive pond described above, such that the eggs traveled further in the pond before settling to the sludge layer. About 90% of the eggs in this pond were Ascaris sp. Helminth egg concentrations in WSP sludge from France have also been reported (Gaspard et al., 1997). Sludge from three ponds was sampled and the egg concentration ranged from 0.56 to 5.7 eggs/g dry solids. In one pond, samples were taken at three locations and the concentration in the middle was more than double that near the inlet and outlet. The viability of the eggs in the WSP sludge was not reported. The low concentration of eggs in these ponds is a reflection of the low prevalence of helminth infections in France. In sludge from 89 treatment plants (most of these were not WSP), the most common species of helminth egg found was from the dog roundworm Toxocara; less than 25% of the eggs were Ascaris sp. 90 1 The most common helminth eggs in wastewater are nematodes. Although none of these studies quantified the inactivation rate of helminth eggs in the WSP sludge, their inactivation has been studied in other similar environments. The inacti- vation of Ascaris eggs, in addition to total and fecal coliform bacteria, fecal streptococci, Salmonella, and poliovirus was studied in sludge lagoons in the southern United States (Reimers et al., 1989). These lagoons were not used to treat wastewater, rather, to provide further treatment of anaerobically digested sludge from conventional municipal treatment plants. Complete inactivation of Ascaris eggs, which were collected from pig intestines and K.L. Nelson spiked in the sludge, was observed after 12 to 15 months of storage. One of the important factors influencing the inactivation rate of Ascaris eggs is tempera- ture. The relationship between temperature and the time to achieve inactivation has been illustrated by compiling data from a wide variety of environments (Feachem et al., 1983), with the required treatment time ranging from more than a year at temperatures below 40°C to several minutes above 65°C. In the sludge lagoon study in the southern U.S., the sludge temperature was around 26°C during the summer months and ranged from 14 to19°C dur- ing the winter months. Although the sludge temperature for the experimental pond in Brazil was not reported, air temperatures in northeast Brazil are frequently above 30°C, so a high- er inactivation rate would be expected. In addition to temperature, compounds present in sludge may affect the inactivation of Ascaris eggs, including ammonia, organic acids, aldehydes, and alcohols, but little is known about their effective concentrations (Ghiglietti et al., 1996; Reimers et al., 2001). As with helminth eggs, the concentrations of pathogenic bacteria, viruses, and protozoa in WSP sludge are expected to vary depending on prevalence in the community and the removal efficiency of the particular pond, but reports on these pathogens are few. The concentration of fecal coliform bacteria in sludge from four ponds in the United States (in Utah and Alaska) ranged from 4.1 × 104 to 2.5 × 105 No./100 mL (Schneiter et al., 1984). In the three ponds in SE Brazil discussed above, the concentrations of fecal coliform bacteria were reported to be 104, 103, and 106 MPN/g TS (Franci, 1999). The most in-depth study of WSP sludge characteristics was undertaken by Carré and Baron (1987).