Environ. Sci. Technol. 2005, 39, 7909-7914
Inactivation of Ascaris suum Eggs by Ammonia BRIAN M. PECSON AND KARA L. NELSON* Department of Civil and Environmental Engineering #1710, University of California, Berkeley, California 94720-1710
Uncharged ammonia is known to cause inactivation of a number of wastewater pathogens, but its effect on Ascaris eggs has never been isolated or quantified. The objectives of this research were to determine the conditions under which ammonia inactivates eggs of the swine Ascaris species, Ascaris suum, and to quantify the impact of ammonia on the U.S. EPA’s requirements for alkaline treatment to produce Class A sludge. Eggs were incubated in controlled, laboratory solutions such that the effects of ammonia concentration and speciation, pH, and temperature could be separated. With a 24-h incubation, the inactivation at all pH levels (range 7-11) was not statistically different in the absence of ammonia. The presence of ammonia (01000 ppm as N) significantly increased Ascaris egg inactivation at pH 9 and 11, and the ovicidal effect was directly related to the concentration of the uncharged NH3 species. Increasing temperatures (32-52 °C) caused increased inactivation at all pH levels and ammonia concentrations. The current EPA treatment requirements to produce Class A biosolids by alkaline treatment have temperature, pH, and time requirements, but do not account for the effect of differences in ammonia concentration on inactivation. To illustrate the potential savings in temperature and pH that could be achieved when accounting for ammonia inactivation, the combinations of ammonia concentration, temperature, and pH needed to achieve 99% inactivation after 72 h were determined. The presence of ammonia at concentrations encountered in sludges and feces (up to 8000 ppm as N) allowed for 99% egg inactivation to be achieved at temperatures up to 14 °C lower than ammonia-free controls. Thus, environmentally relevant concentrations of ammonia may significantly increase the rate of Ascaris egg inactivation during alkaline stabilization.
Introduction Ascaris lumbricoides is a member of the geohelminths, or soil-transmitted helminths, that parasitize the human intestinal tract. It causes the most common parasitic worm infection, ascariasis, affecting nearly 1.4 billion people worldwide (1). Helminth infections such as ascariasis lead to a host of physical and mental disabilities, including cognitive and societal impairment, higher susceptibility to infection, decreased responsiveness to vaccination, and malnutrition (1-3). These disabilities impair the development of several hundred million children in developing countries (1). The infectious cycle is propagated by the ingestion of infective Ascaris eggs, which are widespread in areas with * Corresponding author phone: (510)643-5023; fax: (510)642-7483; e-mail:
[email protected]. 10.1021/es050659a CCC: $30.25 Published on Web 09/09/2005
2005 American Chemical Society
insufficient sanitation. The eggs are highly resistant to desiccation and to a host of chemicals, including strong acids and bases, chlorine, and chloramine (4-6). They can survive in a variety of settings including aerobic and anaerobic environments, within feces, and for up to 7 years in the soil (7-9). The resistance of the Ascaris egg to environmental conditions derives mainly from its eggshell, which has been deemed “one of the most resistant biological structures” (10). Because of their resistance, Ascaris eggs are frequently used as indicators of pathogen inactivation in sludges both for regulatory purposes and for studies of treatment efficiency (11-13). Common methods for inactivating Ascaris eggs in sludges and fecal matter employ high temperatures, high pH, or both. In the absence of other factors, temperatures less than 40 °C are usually not effective at inactivating Ascaris eggs except over very long time periods (>1 y). During biological degradation, such as anaerobic or aerobic digestion, many factors may affect the degree of helminth egg inactivation; however, temperature is still the dominant factor. Thus, at mesophilic temperatures (50 °C) can inactivate eggs to below detectable limits (1416). Ascaris egg inactivation can also be achieved by alkaline stabilization, which is defined here as the storage of sludge or feces at high pH. A number of operating parameters influence the efficiency of alkaline treatment, including pH, temperature, the type and amount of liming material added, and the storage duration. The reported times needed to achieve high levels of inactivation (>95%) vary widely in the literature, ranging from 2 h to 180 d (12, 15, 17-20). The reasons for this variability are often not clear, although differences in the aforementioned operating parameters may explain some of the variation. One factor that is rarely reported, but may also affect alkaline treatment efficiency, is the presence of ammonia. Ammonia is present naturally in wastewater from the hydrolysis of urea and from the degradation of proteins and other nitrogen-containing compounds. Alkaline treatment raises sludge pH and contributes to ammonification. The NH3 species, which dominates at high pH (pKa ) 9.3 at 25 °C), is more lipophilic than NH4+ and can more easily diffuse through biological membranes. NH3 has been shown to cause noxious pH changes within a variety of organisms, including a number of wastewater pathogens (21-23). The effect of various chemicals, including ammonia, on Ascaris egg inactivation was extensively studied in Japan during the first half of the twentieth century (24). Subsequently, Reimers et al. demonstrated that the addition of ammonia to alkalinized sludge increased the inactivation of Ascaris eggs (25). Ghiglietti et al. further investigated Ascaris egg inactivation by alkalinizing with NH4OH instead of lime, the most commonly used material (20, 26). They concluded that alkalinizing with NH4OH was superior after comparing their results to those of Schuh et al., who required 9 weeks to achieve >95% inactivation with lime (17). In both papers by Ghiglietti et al., however, only a low pH control was used, not a high pH control, so the effects of ammonia could not be differentiated from those of pH. Alkaline stabilization has been shown to be an effective, but highly variable, process for the inactivation of Ascaris eggs. Ammonia, a chemical known to inactivate other pathogenic organisms, is found in varying concentrations in sludges and feces and may account for some of this variability. VOL. 39, NO. 20, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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Its effect on Ascaris egg inactivation, however, has not been isolated. Therefore, the first objective of this research was to determine the conditions under which ammonia inactivates the eggs of the swine Ascaris species, Ascaris suum, which is often used as a model for the human species Ascaris lumbricoides. Experiments were conducted under controlled, laboratory conditions such that the effects of ammonia concentration and speciation, pH, and temperature could be separated. The second objective was to quantitatively illustrate how the EPA’s high pH, high-temperature treatment requirements would be impacted when accounting for inactivation by ammonia.
Experimental Section Three sets of experiments were conducted under controlled, laboratory conditions using the same general procedure. Microcentrifuge tubes filled with pH-buffered ammonium chloride solutions and Ascaris suum eggs were incubated at a range of temperatures. The viability of the eggs was determined after every treatment. In the first set of experiments, the concentration of total NH4Cl was held constant at pH 7, 9, and 11 for 1 or 24 h. In the second group of experiments, the importance of ammonia speciation was investigated by maintaining a constant concentration of the uncharged NH3 species (instead of total NH4Cl) at both pH 9 and 11 for 24 h. In the third experiment, the temperatures and ammonia concentrations necessary to achieve 99% inactivation of Ascaris eggs after 72-h incubation were determined for pH 7, 9, and 11. Experimental Procedure. For each experiment, solutions of varying ammonia concentrations were prepared in 60-mL plastic bottles and held at given pH levels with buffered solutions. Stocks of 0.4 M HEPES, CHES, and CAPS were prepared and diluted to 0.1 M to buffer experimental solutions at pH 7, 9, and 11, respectively (all chemicals were purchased from Fisher Scientific, Pittsburgh, PA). An ammonia stock of 10 000 ppm as N was made from NH4Cl (99.9% purity) and kept at low pH to prevent ammonia volatilization. The buffered ammonia solutions were heated to experimental temperatures in a water bath (model 288, Precision, Winchester, VA), and then the pH was adjusted with KOH and HCl (Accumet pH electrode, Fisher Scientific, with model 295 pH/Temperature/mV/ISE Meter, Beckman Coulter Inc., Fullerton, CA). From each 60-mL bottle, three replicates were pipetted into 1.5-mL microcentrifuge tubes that were immediately capped. Care was taken to fill the tubes completely to minimize the headspace into which ammonia could volatilize. Triplicate samples for each pH/ammonia/temperature combination were placed in a covered water bath, and roughly 2000 Ascaris suum eggs were added (Precision Water Bath model 288, Winchester, VA; rated accuracy: (0.1 °C at 37 °C). After the incubation period (1, 24, or 72 h), the pH of each sample was measured, and the tubes were cooled to room temperature on ice and centrifuged at 1500g for 2 min. The supernatant was removed, and the eggs were resuspended in 0.1 M H2SO4. Tubes were placed uncapped in an incubator for 4 weeks under optimal aerobic growing conditions (28 °C and 100% relative humidity, model 3015 Waterjacket Incubator, VWR Scientific Products). After incubation, the eggs were centrifuged at 1500g for 2 min and resuspended in 15% household bleach solution for 20 min to remove the outer layers of the eggs. This decortication step allowed for easier viewing of the developmental stage of the larva under the microscope. After decortication, eggs were rinsed, resuspended in 100 µL of water, and pipetted onto a microscope slide. Eggs were viewed using brightfield microscopy at 100× magnification (Olympus BH-2 microscope). Eggs that had developed to the fully larvated stage were considered viable, while all others were deemed inactivated. The percent of nonviable eggs in the 7910
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TABLE 1. Percentage of Total Ammonia as Uncharged NH3 from 32 to 52 °C at pH 7, 9, and 11, Assuming Infinite Dilution T (°C)
pH 7
pH 9
pH 11
32 36 40 44 48 52
0.9 1.2 1.5 2.0 2.5 3.2
48.0 54.7 61.0 66.8 72.1 76.6
98.9 99.2 99.4 99.5 99.6 99.7
stock egg suspensions was always very low (∼1%). As such, nearly all of the egg inactivation was attributed to the treatment process. The fraction of inactivated eggs was calculated as the number of inactivated eggs divided by the total eggs counted (n ) 200 per sample). Ascaris suum eggs were purchased from Excelsior Sentinel (Ithaca, NY); the company collected the eggs from the intestinal contents of pigs, using sequential sieving to concentrate and clean the eggs. Eggs were shipped at concentrations between 104 and 105/mL and stored at 4 °C in 0.5% formalin. The eggs were not exposed to any other chemicals prior to the experiments. Four different batches of eggs were used for these experiments. Experiment 1: Constant Total NH4Cl at pH 7, 9, and 11. Eggs were incubated for 1 and 24 h at varying temperatures (32-52 °C) and NH4Cl concentrations (0, 10, 100, and 1000 ppm as N) at pH 7, 9, and 11. The activity of un-ionized ammonia varied as a function of temperature, pH, and ionic strength according to the Arrhenius, Henderson-Hasselbach, and Davies’ equations, respectively (27, 28):
KNH4+|T2 ) KNH4+|T1 × exp [NH3] ) NTOT ×
(
( (
∆H hR 1 1 R T1 T2
KNH4+
KNH4+ + [H+]
)
))
log γNH3 ) -Azi2[(xI/(1 + xI)) - 0.2I] {NH3} ) γNH3 × [NH3] where KNH4+ is the acid dissociation constant for NH4+, NTOT ) [NH4+] + [NH3], γ is the activity coefficient, I is the ionic strength of the solution, and {NH3} is the un-ionized ammonia activity. The percentage of NH4Cl in the un-ionized NH3 form for the three pH levels is shown in Table 1. Increasing the temperature decreases the pKa, which causes significant changes in the fraction of un-ionized ammonia at pH levels near the pKa (see pH 9 in Table 1). A more detailed investigation of the ammonia effect was conducted at pH 9 and 11, with various NH4Cl levels (0, 100, 200, 300, 400, 500, and 1000 ppm as N) at 40-48 °C. These experiments allowed for the isolation and characterization of the response of Ascaris eggs to each of the operating parameters (pH, temperature, and exposure period) in the absence and presence of ammonia. Most importantly, by being able to compare the ammonia effect across a pH range at a given NH4Cl concentration, and across a range of NH4Cl concentrations for a given pH, the effect of pH could be separated from that of ammonia. Experiment 2: Constant NH3 at pH 9 and 11. Eggs were exposed to solutions of equivalent un-ionized NH3 activity (0, 0.011, 0.017, 0.022, 0.027, 0.033, 0.038, 0.054) at both pH 9 and 11 for 24 h at 44 °C. Different amounts of total NH4Cl were needed to achieve a given NH3 activity at the two pH levels (Table 2). Activity is reported instead of concentration
TABLE 2. Concentration of NH4Cl (ppm as N) Needed To Achieve Given Levels of NH3 Activity at 44 °C for pH 9 and 11 NH3 activity
pH 9
pH 11
0 0.011 0.017 0.022 0.027 0.033 0.038 0.054
0 299 449 598 748 898 1047 1496
0 200 300 400 500 600 700 1000
because it takes into account the effect of ionic strength on the chemical reactivity of the species and, thus, may be more important biologically. Experiment 3: 99% Inactivation after 72-h Incubation. The minimum concentration of NH4Cl needed to cause 99% inactivation (average for three replicates) after 72-h incubation was determined for pH 7, 9, and 11 for a range of temperatures (32-52 °C). The NH4Cl concentrations ranged from 0 to 8000 ppm as N in 500 ppm intervals. To further minimize variability due to ammonia volatilization, screwtop microcentrifuge tubes with silicone O-rings were used. In addition, 1-mL samples of solution were collected before and after treatment, and the ammonia concentration was verified with an ion-specific electrode (Beckman Instruments, Inc., Fullerton, CA). Statistical Analysis. All statistical analyses of data were performed with JMP Statistical Software (SAS Institute Inc., Cary, NC).
Results Experiment 1: Constant Total NH4Cl at pH 7, 9, and 11. The results from incubating Ascaris in the absence of ammonia for 1 and 24 h are presented in Figure 1. At both exposure periods, a threshold temperature range occurred where the eggs went from a baseline level of inactivation to >99% inactivation. At 1 h, the threshold range occurred between 45 and 51 °C, and, as the exposure time increased to 24 h, this range dropped to 40-48 °C. The threshold temperature range may have been narrower if more temperatures had been studied. In the absence of ammonia, there was no significant difference in inactivation between the three pH levels at either of the treatment durations (analysis of variance, R ) 0.05). Inactivation in control samples verified that the buffers themselves exerted no inactivating effect (data not shown). A 1-h incubation in the presence of ammonia did not cause any distinguishable effect on inactivation (data not shown). A pattern did emerge, however, after the 24-h incubation with ammonia. In the presence of 10 and 100 ppm NH4Cl-N, the inactivation curves at all three pH levels were not significantly different from the ammonia-free control (analysis of variance with Tukey’s test, R ) 0.05, data not shown). With 1000 ppm NH4Cl-N, however, the pH 9 and 11 solutions had significantly greater inactivation at 44 °C (Figure 2). The pH 7 ammonia solutions were not statistically different from the control at any temperature (36-48 °C) or ammonia concentration, even at 1000 ppm. The next set of experiments focused on pH 9 and 11, over the same range of added NH4Cl (0-1000 ppm as N), at the 24-h threshold temperature range (40-48 °C) where the ammonia effect had been demonstrated (Figure 3). Inactivation jumped sharply from baseline levels to complete inactivation over a 6 °C range (41-47 °C). Inactivation at pH 11 was significantly greater than that at pH 9 except when the added NH4Cl concentrations were low (e400 ppm for T e 43 °C, and e100 ppm for 44 °C e T e 46 °C; analysis of
FIGURE 1. Ascaris egg inactivation as a function of temperature after 1- and 24-h incubation in the absence of ammonia. Symbols: pH 7, ×; pH 9, ]; pH 11, O.
FIGURE 2. Ascaris egg inactivation as a function of temperature after 24-h incubation in 1000 ppm NH4Cl as N. Symbols: 0 ppm pH 7 control, 0; 1000 ppm solutions at pH 7, ×; pH 9, ]; pH 11, O.
FIGURE 3. Ascaris egg inactivation as a function of total NH4Cl (ppm as N) and temperature after 24-h incubation at pH 9 and 11. Symbols: pH 9, gray walls; pH 11, white walls. variance, R ) 0.05). At T > 46 °C, the effect of ammonia could not be distinguished because the effect of heat alone inactivated >99% of the eggs. Experiment 2: Constant NH3 at pH 9 and 11. At NH3 activities from 0 to 0.054, the fraction of inactivated eggs at pH 9 and 11 was not statistically different at 44 °C (Figure 4). Experiment 3: 99% Inactivation at 72 h. The specific combinations of temperature and NH4Cl leading to 99% inactivation after 72-h incubation are shown for the three VOL. 39, NO. 20, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 4. Ascaris egg inactivation as a function of uncharged NH3 activity after 24-h incubation at 44 °C. Symbols: pH 9, ]; pH 11, O.
FIGURE 5. Ammonia, temperature, and pH combinations resulting in 99% inactivation of Ascaris eggs after 72-h incubation. Symbols: pH 7, ×; pH 9, ]; pH 11, O. pH levels in Figure 5. All data points represent average inactivations of at least 99% measured in three replicates. In the absence of ammonia, a minimum temperature of 48 °C was needed for 99% inactivation at all pH levels. Increasing NH4Cl did not lower the temperature requirements for pH 7, but led to progressively larger decreases in temperature for pH 9 and 11, respectively. For example, at an NH4Cl level of 2500 ppm as N, an increase of pH from 7 to 9 decreased the temperature requirement from 48 to 41 °C. Raising the same concentration from pH 7 to 11 decreased the temperature requirement by 10 °C, to 38 °C. Within the range of NH4Cl tested (0-8000 ppm as N), the largest decreases in temperature were 12 °C at pH 9 and 14 °C at pH 11.
Discussion At every exposure period tested, the increased inactivation with increasing temperature was consistent with previously published research (4, 8, 29). Interestingly, pH changes alone did not cause increased inactivation at the 1- and 24-h incubation periods, nor did they alter the minimum temperature needed for 99% inactivation after 72-h incubation (see Figure 5). At every time period tested, the pH did affect inactivation when NH4Cl was added, with higher levels of inactivation occurring with increasing pH (Figures 2, 3, and 5). The effect of pH, however, was indirect. Changes in pH affected the fraction of total ammonia in the uncharged NH3 form, the form that was responsible for ammonia inactivation of Ascaris eggs. Indeed, as long as the uncharged NH3 activity was held constant, the inactivation levels were also constant, regardless of pH (Figure 4). A minimum NH3 concentration was necessary to cause ammonia inactivation at a given temperature. Below this concentration, solutions at both pH 9 and 11 had levels of inactivation that were equivalent to each other and to the 7912
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ammonia-free control (e.g., Figure 3 at temperatures 41-43 °C with NH4Cl concentrations of 99% at pH 11 vs ∼66% at pH 9). For example, with a NH4Cl concentration of 2500 ppm as N, the pH 9 sample required 41°C for 99% inactivation, while pH 11 needed only 38 °C. This increase in ammonia inactivation with increasing pH would be expected to level out as ammonia speciation moves asymptotically toward 100% deprotonation. For example, raising the pH from 11 to 12 would convert an additional amount of NH4+ that is equivalent to less than 1% of the total. The concentrations of ammonia used throughout these experiments were chosen to represent the concentrations that might naturally be found in wastewater sludges, fecal sludges, and agricultural slurries (34-38). Differing concentrations occur for numerous reasons, including the various sources of wastewater (e.g., low-strength, dilute municipal wastewater vs highly concentrated urine and feces of agricultural wastes), the type of biosolids treatment employed (e.g., aerobic vs anaerobic), and the process of sludge dewatering (39). The results of this research may help to make sense of the previously published data: differences in ammonia concentrations could help to explain how two sludges with identical pH levels and temperatures exhibit significant differences in inactivation times (12, 15, 17-20). Ghiglietti et al. reported increased Ascaris egg inactivation with a high pH, NH4OH solution, and the results of our research suggest that the increase was not due to the pH change alone, but to the presence of NH3. The results obtained by Ghiglietti et al. on the degree of inactivation are in agreement with those reported here. It may be possible to increase pathogen inactivation during alkaline stabilization by making modifications to optimize inactivation by ammonia. For example, the practice of covering and insulating alkaline storage tanks prevents ammonia loss by volatilization and conserves the heat produced during quicklime (CaO) addition. High levels of inactivation were achieved in our studies within the range of temperatures achieved by typical quicklime dosing (3040 °C). It is, therefore, possible that the heat produced by quicklime addition, coupled with ammonia inactivation, may be sufficient for the rapid inactivation of Ascaris without the need for additional heating. This method may be a low-cost treatment option for producing Class A sludges. The agricultural value of treated sludge may also be increased by accounting for and optimizing ammonia inactivation. The loading rate of sludge onto agricultural fields is often limited by the dry weight of the material. The addition of large amounts of lime increases the dry mass of treated sludge, thereby decreasing the amount that can be applied to a given field. The high pH level currently required (pH 12) also limits the application of sludges to fields with acidic
soils. Interestingly, ammonia inactivation was still capable of causing 99% inactivation when the pH was raised to just 9. By decreasing the lime dose to raise the pH to 9 instead of 12, the loading rates of sludge onto agricultural areas would increase, the cost of liming would decrease, and a potentially larger geographic area could accept this lower pH sludge onto their fields. Most importantly, these benefits could be realized without compromising the quality of pathogen inactivation. One implication, however, is that the liming process would no longer fulfill the vector reduction requirements, which require achieving a minimum of pH 12. Further research is needed to quantify the inactivating effect of ammonia in actual sludges. In the presence of organic-rich sludge, ammonia may undergo reactions such as ion exchange that make it less available for Ascaris inactivation. Other compounds besides ammonia may also play a role in pathogen inactivation, including organic acids, aldehydes, and alcohols (31). Once these various factors are quantified, the approach illustrated in Figure 5 could be used to determine the necessary treatment conditions for producing Class A sludges.
Acknowledgments We thank the University of California, Berkeley division of the Academic Senate Committee on Research, for awarding a Junior Faculty Research Grant to provide support for this research.
Literature Cited (1) Crompton, D. W. T. Ascaris and ascariasis. In Advances in Parasitology; Academic Press: New York, 2001; Vol. 48, pp 285375. (2) Moreira-Silva, S. F.; Leite, A. L. A.; Brito, E. F.; Pereira, F. E. L. Nematode infections are risk factors for staphylococcal infection in children. Mem. Inst. Oswaldo Cruz 2002, 97, 395-399. (3) Cooper, P. J.; Chico, M.; Sandoval, C.; Espinel, I.; Guevara, A.; Levine, M. M.; Griffin, G. E.; Nutman, T. B. Human infection with Ascaris lumbricoides is associated with suppression of the interleukin-2 response to recombinant cholera toxin B subunit following vaccination with the live oral cholera vaccine CVD 103-HgR. Infect. Immun. 2001, 69, 1574-1580. (4) Barrett, J. Studies on induction of permeability in Ascaris lumbricoides eggs. Parasitology 1976, 73, 109-121. (5) Krishnaswami, S. K.; Post, F. J. Effect of chlorine on Ascaris (Nematoda) eggs. Health Lab. Sci. 1968, 5, 225-232. (6) Wharton, D. A. The production and functional morphology of helminth egg-shells. Parasitology 1983, 86, 85-87. (7) Reimers, R. S.; McDonell, D. B.; Little, M. D.; Bowman, D. D.; Englande, A. J.; Henriques, W. D. Effectiveness of wastewater sludge treatment processes to inactivate parasites. Water Sci. Technol. 1986, 18, 397-404. (8) Feachem, R. G.; Bradley, D. J.; Garelick, H.; Mara, D. D. Sanitation and Disease: Health Aspects of Excreta and Wastewater Management; John Wiley and Sons: New York, 1983. (9) Gaasenbeek, C. P. H.; Borgsteede, F. H. M. Studies on the survival of Ascaris suum eggs under laboratory and simulated field conditions. Vet. Parasitol. 1998, 75, 227-234. (10) Wharton, D. Nematode egg-shells. Parasitology 1980, 81, 447463. (11) U.S. EPA. “A Plain English Guide to the EPA, Part 503 Biosolids Rule,” 1994. (12) Brewster, J.; Oleszkiewicz, J.; Bujoczek, G.; Reimers, R. S.; AbuOrf, M.; Bowman, D.; Fogarty, E. Inactivation of Ascaris suum eggs in digested and dewatered biosolids with lime and fly ash at bench scale and full scale. J. Environ. Eng. Sci. 2003, 2, 395400. (13) Capizzi, S.; Schwartzbrod, J. Surface properties of Ascaris suum eggs: hydrophobic potential and Lewis acid-base interactions. Colloids Surf., B 2001, 22, 99-105. (14) Carrington, E. G.; Pike, E. B.; Auty, D.; Morris, R. Destruction of fecal bacteria, enteroviruses and ova of parasites in wastewater sludge by aerobic thermophilic and anaerobic mesophilic digestion. Water Sci. Technol. 1991, 24, 377-380. (15) Gantzer, C.; Gaspard, P.; Galvez, L.; Huyard, A.; Dumouthier, N.; Schwartzbrod, J. Monitoring of bacterial and parasitological contamination during various treatment of sludge. Water Res. 2001, 35, 3763-3770. VOL. 39, NO. 20, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
7913
(16) Plym-Forshell, L. Survival of Salmonellas and Ascaris suum eggs in a thermophilic biogas plant. Acta Vet. Scand. 1995, 36, 7985. (17) Schuh, R.; Philipp, W.; Strauch, D. In Inactivation of microorganisms in sewage sludge by stabilization processes; Strauch, D., Havelaar, A. H., L’Hermite, P., Eds.; Elsevier Applied Science Publishers: London and New York; Hohenheim, Germany, 1984; pp 100-110. (18) Mendez, J. M.; Jimenez, B. E.; Barrios, J. A. Improved alkaline stabilization of municipal wastewater sludge. Water Sci. Technol. 2002, 46, 139-146. (19) Eriksen, L.; Andreasen, P.; Ilsoe, B. Inactivation of Ascaris suum eggs during storage in lime treated sewage sludge. Water Res. 1996, 30, 1026-1029. (20) Ghiglietti, R.; Genchi, C.; DiMatteo, L.; Calcaterra, E.; Colombi, A. Survival of Ascaris suum eggs in ammonia-treated wastewater sludges. Bioresour. Technol. 1997, 59, 195-198. (21) Warren, K. S. Ammonia toxicity and pH. Nature 1962, 195, 4749. (22) Cramer, W. N.; Burge, W. D.; Kawata, K. Kinetics of virus inactivation by ammonia. Appl. Environ. Microbiol. 1983, 45, 760-765. (23) Jenkins, M. B.; Bowman, D. D.; Ghiorse, W. C. Inactivation of Cryptosporidium parvum oocysts by ammonia. Appl. Environ. Microbiol. 1998, 64, 784-788. (24) Morishita, K., Komiya, Y., Matsubayashi, H., Eds. Progress of medical parasitology in Japan; Meguro Parasitological Museum: Tokyo, 1972; Vol. IV. (25) Reimers, R. S.; Little, M. D.; Englande, A. J.; McDonell, D. B.; Bowman, D. D.; Hughes, J. M. “Investigation of parasites in sludges and disinfection techniques,” U.S. Environmental Protection Agency, 1985. (26) Ghiglietti, R.; Rossi, P.; Ramsan, M.; Colombi, A. Viability of Ascaris suum, Ascaris lumbricoides, and Trichuris muris eggs to alkaline pH and different temperatures. Parassitologia 1995, 37, 229-232. (27) Benjamin, M. M. Water Chemistry; McGraw-Hill: Boston, 2002. (28) Pankow, J. F. Aquatic Chemistry Concepts; Lewis Publishers: Chelsea, MI, 1991. (29) Aitken, M. D.; Sobsey, M. D.; Blauth, K. E.; Shehee, M.; Crunk, P. L.; Walters, G. W. Inactivation of Ascaris suum and poliovirus
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(30)
(31)
(32)
(33)
(34)
(35)
(36)
(37) (38)
(39)
in biosolids under thermophilic anaerobic digestion conditions. Environ. Sci. Technol. 2005, 39 (15), 5804-5809. Fairbairn, D.; Passey, B. I. The lipid components in the vitelline membrane of Ascaris lumbricoides eggs. Can. J. Biochem. Physiol. 1955, 33, 130-134. Reimers, R. S.; Bowman, D. D.; Schafer, P. L.; Tata, P.; Leftwich, B.; Atique, M. M. Factors Affecting Lagoon Storage Disinfection of Biosolids. WEF/AWWA/CWEA Joint Residual and Biosolids Management Conference Biosolids 2001 2001. SEMARNAT. “NOM-004-SEMARNAT-2002. Proteccion ambiental. Lodos y biosolidos - especificaciones y limites maximos permisibles de contaminantes para su aprovechamiento y disposicion final,” 2003. Jimenez, B.; Mendez, J. I.; Barrios, J. A.; Salgado, G.; Sheinbaum, C. Characterization and evaluation of potential reuse options for wastewater sludge and combined sewer system sediments in Mexico. Water Sci. Technol. 2004, 49, 171-178. Misselbrook, T. H.; Pain, B. F.; Headon, D. M. Estimates of ammonia emission from dairy cow collecting yards. J. Agric. Eng. Res. 1998, 71, 127-135. Sutton, A. L.; Kephart, K. B.; Verstegen, M. W. A.; Canh, T. T.; Hobbs, P. J. Potential for reduction of odorous compounds in swine manure through diet modification. J. Anim. Sci. 1999, 77, 430-439. Sorensen, P.; Fernandez, J. A. Dietary effects on the composition of pig slurry and on the plant utilization of pig slurry nitrogen. J. Agric. Sci. 2003, 140, 343-355. Epstein, E. Land Application of Sewage Sludge and Biosolids; Lewis Publishers: Boca Raton, FL, 2002. Lue-Hing, C. Municipal Sewage Sludge Management: A Reference Text on Processing, Utilization, and Disposal, 2nd ed.; Technomic Pub. Co.: Lancaster, PA, 1998. McFarland, M. J. Biosolids Engineering; McGraw-Hill: New York, 2001.
Received for review April 6, 2005. Revised manuscript received July 15, 2005. Accepted August 1, 2005. ES050659A
