Concentrations and Inactivation of Ascaris Eggs and Pathogen

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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 Publishing 2003 © IWA 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 concentrated 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 distribution of eggs was studied by collecting samples from eight different locations throughout 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 facultative 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 inactivation 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 temperature. 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 during 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). Stratified sludge cores were collected near the inlet and outlet of a primary pond that had been operating for 10 years, and various physical, chemical, and micro- biological parameters were measured. The concentrations of total and fecal coliform bacteria and fecal streptococci were found to decrease with depth in the sludge layer. Maximum concentrations around 107 MPN/100 mL were found at the sludge surface near the inlet, and were lower near the outlet by approximately one log. The concentrations of fecal coliform bacteria and fecal streptococci were lower by 3 to 4 logs in the deepest sludge compared to the surface sludge. These findings suggest that significant inactivation of pathogens occurred as the sludge aged.

Recent experimental ﬁndings The goal of our recent experiments was to determine the concentrations and inactivation rates of Ascaris eggs and several indicator organisms in the sludge layer of three Mexican primary wastewater stabilization ponds – two facultative and one anaerobic (Nelson et al., 2002; Nelson and Darby, 2002). Estimating the inactivation rate of organisms in the sludge layer of WSPs is not straightforward, however, and methods to collect such information had not been validated previously. Therefore, we used two independent experimental methods for each organism – sludge cores and either dialysis chambers or a batch test. The sludge cores were collected from 3 to 7 locations in each pond, the age of the sludge at each depth was estimated, and the inactivation rate of each organism was determined from changes in concentration with sludge age. Helminth eggs and fecal coliform bacteria were measured in the cores from all three ponds, and in one pond fecal enterococci and somatic 91 and F+ coliphages were also evaluated. For the second measure of Ascaris egg inactivation, dialysis chambers loaded with Ascaris eggs were stored in the sludge layer of one of the ponds for 14 months and 3 chambers were removed approximately once a month. The inactivation rate of the eggs was determined from a decrease in the percentage of viable eggs recovered from the chambers. For the second measure of indicator organism inactivation, a batch of sludge was removed from one of the ponds and stored in the laboratory at

K.L. Nelson approximately the same temperature as the pond’s sludge layer. The concentrations of fecal coliform bacteria, fecal enterococci, F+ coliphage, and somatic coliphage were measured periodically for 7 months and the inactivation rates were determined from the decrease in concentration with time. First-order kinetics were found to adequately describe the inactivation of all the organisms studied. The rate constants determined for all of the organisms and from the various methods are summarized in Table 1. The average first-order inactivation rate constants for Ascaris eggs in all three ponds were similar, ranging from 0.0007 to 0.001 d–1. In the Texcoco pond, we compared the apparent inactivation of Ascaris eggs in the sludge cores with that observed in dialysis chambers. Over the time period of the dialysis chamber experiment (14 months), the rate constants determined by the two methods were similar, around 0.002 d–1. However, the data from the sludge cores indicated that the inactivation rate of Ascaris eggs decreased significantly after this time, resulting in a lower average long-term inactivation rate. Whether the inactivation rate truly decreased after 14 months, or whether the apparent tailing was caused by contamination of the older sludge by the newer sludge could not be determined. It is concluded, therefore, that a first-order rate constant of 0.001 d–1 is a conservative value for estimating the average inactivation of Ascaris eggs in the sludge layer of WSPs in central Mexico. More than 85% of the helminth eggs isolated from the sludge core samples were Ascaris sp.; the remaining eggs were Trichuris, Hymenolepis, and Toxocara sp. Although the concentrations of Trichuris, Hymenolepis, and Toxocara were too low to permit determination of their inactivation rate, Ascaris eggs appeared to be more resistant and therefore an appropriate indicator for the others. (In addition, the viability of Hymenolepis could not be determined by the method used.) The rate constants calculated from both the sludge cores and the batch experiment for fecal coliform, fecal enterococci, F+ coliphage, and somatic coliphage were in close agreement (Table 1). It is concluded that values of 0.1, 0.01, and 0.001 d–1 are conservative estimates of the first order rate constants for fecal coliform (and fecal enterococci), F+ coliphage, and somatic coliphage, respectively, under the conditions of our research.

Table 1 First-order rate constants for the inactivation of Ascaris eggs and indicator organisms. The inactivation rate of each was determined by two different methods – sludge cores and dialysis chambers (Ascaris eggs) or sludge cores and a batch test (indicator organisms )

k, d–1

Organism Sludge cores Batch test Dialysis chambers

Ascaris eggs Mexicaltzingo 0.0009 Texcoco 0.0007a 0.0021 Xalostoc 0.0010 Indicator organisms (Xalostoc) Somatic coliphage 0.0016 0.0074 F+ coliphage 0.016 0.037 Fecal coliform 0.13 0.16 Fecal enterococci 0.26 0.20 a Over the ﬁrst 16 months (the duration of the dialysis chamber experiment), the inactivation rate was calcu- 92 lated to be 0.0019 d–1 For all the organisms studied, the inactivation rates calculated based on the two independent methods showed a high level of agreement. However, the quality and quantity of information provided by each method was different. The main advantage of the sludge core method was that information could be collected on the entire operational period of the pond (5 to 15 years for the ponds studied in this research) in a single sampling occasion. In con- trast, the dialysis chambers (for Ascaris eggs) and batch test (for fecal coliform, fecal enterococci, F+ coliphage, and somatic coliphage) experiments were carried out for 7 to 16 K.L. Nelson months, and even over these time periods complete inactivation was not always observed. Because the inactivation rates were sometimes observed to decrease with time, however, it is recommended that inactivation rates calculated using dialysis chambers or a batch test not be applied to time periods longer than that studied (unless complete inactivation is observed). The dialysis chamber and batch tests, however, provided more detailed information than the sludge cores. The main disadvantages of the sludge core method are the potential for contamination during sampling (of older sludge by newer sludge) and uncertainty about the depositional history of the sludge layer. Nevertheless, in many situations sludge cores may provide the most efficient method for gathering long-term data on pathogen concentrations and inactivation rates in the WSP sludge. The second emphasis of our work was to determine the concentration and distribution of organisms in the sludge layer. In all ponds, the concentrations of helminth eggs and indicator organisms varied significantly with location in the pond. In both facultative ponds, the concentration of total helminth eggs (all species, viable and non-viable) was highest near the pond inlet and decreased substantially with distance from the inlet. In the anaerobic pond, however, the concentration increased slightly from the inlet towards the outlet. We believe these profiles are representative of the ponds’ hydraulics; because the facultative ponds had long detention times (11 and 47 d), the overflow rates were low and sedimentation of the eggs occurred near the inlet, whereas the short detention time in the anaerobic pond (2.5 d) created a high overflow rate and eggs settled out throughout the entire pond. The concentration of helminth eggs was consistent with the sludge distribution in the ponds (Nelson et al., 2002) – high egg concentrations were found where the depth of accumulated sludge was greatest. Although the concentrations of fecal coliform bacteria and the other indicator organisms varied with pond location, no trends were observed to explain the variation. To summarize, we observed large variations in the concentrations of all the organisms in both horizontal and vertical directions. Thus, there are now several reports on the hetero- geneous distribution of pathogens and indicator organisms in WSPs (Ayres et al., 1993; Carré and Baron, 1987; Franci, 1999; Gaspard et al., 1997; Nelson et al., 2002). These findings have important implications for sludge monitoring and characterization, i.e., sampling efforts must recognize that WSP sludge cannot be characterized by collecting grab samples from just one or two locations. It is essential that efforts to measure pathogen concentrations in the sludge layer incorporate the collection of complete sludge cores (to capture vertical heterogeneity) from several representative locations throughout the pond (to capture horizontal heterogeneity). Utilizing this approach, the concentrations of helminth eggs and indicator organisms in the sludge layers of the three ponds are reported in Table 2. Both the mean and maximum concentrations were calculated, because both are useful for assessing the potential risk associated with removing the sludge. The value that should be used depends, in part, on the removal method used. At this time we are not aware of any governmental regulations that specify how samples should be collected from WSP sludge. The experimental values from the Mexican ponds are compared with the biosolids regulations on helminth eggs and fecal coliforms stipulated by the USEPA and the Mexican government in Table 2. None of the 93 Table 2 Meana and maximum concentrations of helminth eggs and indicator organisms in the sludge layers of the three ponds and maximum values of helminth eggs and fecal coliform bacteria allowed by the U.S. EPAb and Mexican governmentc in biosolids that are to be land applied

Total helminth Viable helminth F+ coliphage, Fecal coliform eggs, eggs/g TS eggs, eggs/g TS pfu/g TS bacteria, MPN/g TS

Location Mean Max. Mean Max. Mean Max. Mean Max.

K.L. Nelson THIS RESEARCH Mexicaltzingo 129 184 25 55 1.3 × 105 1.2 × 107 Texcoco 49 273 25 169 5.7 × 104 1.5 × 107 Xalostoc 277 657 48 257 1.2 × 104 1.3 × 106 3.1 × 104 4.4 × 107

REGULATIONd U.S. EPA Class A 0.25e 1 × 103 U.S. EPA Class B no limit 1 × 103 Mexico Class A 10 1 × 103 Mexico Class B 35 2 × 106 a For helminth eggs the arithmetic mean was calculated, whereas the geometric mean was calculated for the indicator organisms b U.S. EPA (1993) c INE (2000). Also stipulates a Salmonella concentration ≤ 3 and ≤ 300 MPN/g TS for Class A and B biosolids, respectively d No regulations exist for the concentration of Somatic coliphage, F+ coliphage, or Fecal enterococci e The actual regulation stipulates < 1 egg/4 g TS.

mean values in the WSP sludges complied with the USEPA standards for Class A or Class B biosolids. However, sludge from the Mexicaltzingo and Texcoco ponds complied with Mexican standards for Class B biosolids (< 35 eggs/g total solids and < 2 × 106 MPN/g total solids). The average concentration of viable helminth eggs in the sludge from Xalostoc (48 eggs/g total solids) exceeded the value allowed for reuse or disposal of biosolids in Mexico. Based on the current regulations and the mean concentrations observed, the sludge from the Xalostoc pond would require further treatment upon removal. Treatment options include, but are not limited to, alkaline (lime) treatment, sludge drying beds, and aerobic or anaerobic thermophilic sludge digestion.

Conclusions Using a range of methods, we have estimated the inactivation rates of fecal coliform bacteria, fecal enterococci, F+ coliphage, somatic coliphage, and Ascaris eggs in WSP sludge in central Mexico. Although no values on inactivation rates were found in the literature for comparison, our results are generally consistent with previous reports on the relative inactivation rates of these organisms, namely that the rate of inactivation (roughly) decreases in the order in which the organisms are listed above. It is concluded that many pathogenic enteric bacteria would be inactivated within several months, whereas the inactivation of enteric viruses may take several years, depending on the initial concentrations. About 50% of Ascaris eggs were inactivated within the first year. Previous studies on the inactivation of Ascaris eggs and indicator organisms in other environments have shown temperature to significantly affect the inactivation rate, thus, the inactivation rates measured in central Mexico may be considerably different from those in WSPs located in regions with different climates. One challenge with removing sludge from continuously operating WSPs is that some fresh sludge will always be present. Because the microorganisms were inactivated with time, the new sludge exerted a dominant influence on the average concentration of organ- 94 isms in the sludge layer. Furthermore, the inactivation rate of all organisms decreased with time; therefore, the greatest benefit (in terms of pathogen inactivation) occurred during the first period of storage. The main implication of this finding is that substantial benefit may be achieved by allowing a pond to remain out of service for some time before removing its sludge. The necessary time to reduce the pathogen concentrations to safe levels depends, of course, on the initial concentrations in the new sludge and the inactivation rates. Using the sludge core method outlined in our research, these values can be determined for any organism and any pond. K.L. Nelson

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