APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1992, p. 3514-3516 0099-2240/92/113514-03$02.00/0 Copyright © 1992, American Society for Microbiology
Vol. 58, No. 11
Efficacy of Activated Sludge in Removing Cryptosporidium parvum Oocysts from Sewage I. VILLACORTA-MARTINEZ
DE MATURANA, M. E. ARES-MAZAS,* AND M. J. LORENZO-LORENZO
D. DURAN-OREIRO,
Laboratory of Parasitology, Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Santiago de Compostela, 15706 Santiago de Compostela, La Corufia, Spain Received 25 February 1992/Accepted 1 September 1992
Primary clarifier effluent (procedure B) and final effluent (procedure A) from a wastewater treatment plant enriched with Cryptosporidium parvum oocysts obtained from the feces of naturally infected calves. Procedure B samples alone were subjected to a laboratory simulation of activated-sludge treatment. Coccidium-free neonatal CD-1 mice were then inoculated intragastrically with procedure A or procedure B samples. Seven days after inoculation, the intensity of oocyst infection in procedure B mice was 91% less than in procedure A mice (controls).
were
Cryptosporidium parvum (Apicomplexa, Cryptosporidiidae) is a monoxenous coccidian protozoan parasite causing diarrheal disease in a wide range of vertebrates including humans, with serious consequences in neonates and immunodeficient individuals. Recent reports of cryptosporidiosis outbreaks suggest that contamination of water supplies plays an important role in transmitting the disease (3-5, 8, 16), a hypothesis supported by the detection of oocysts of both human and animal origin in surface waters and sewage (11-13, 15). Furthermore, it has been shown that disinfectants for drinking water purification, with the possible exception of ozone (9, 14), are not very effective against C. parvum. In this study, we evaluated the efficacy of a sewage treatment system (activated sludge and flocculation) in removing C. parvum oocysts from contaminated sewage by using a laboratory simulation of the treatment process. MATERIALS AND METHODS Animals and husbandry. The 12 litters of coccidium-free neonatal CD-1 mice used were kept separately with their dams at 20°C in Panlab plastic cages with wire mesh tops and wood shavings for bedding and received a commercial pelleted feed (Letica, Barcelona, Spain) and water ad libitum.
Oocysts. One- to two-week-old Friesian calves were individually screened for excretion of C parvum oocysts by carbolfuchsin staining (6). Fresh fecal material was collected by rectal sampling, immediately suspended 1:1 in cold 5% (wt/vol) aqueous potassium dichromate solution, and stored at 4°C until use. After several washes with demineralized water to eliminate potassium dichromate, the feces were washed through a 45-,um-pore-size mesh and centrifuged several times in demineralized water-diethyl ether at 1,000 x g for 5 min; the resulting concentrated fluid was washed again to eliminate the diethyl ether. For counting, 0.2 ml of the suspension was mixed with 0.8 ml of a malachite green preparation (malachite green, 0.16 g; sodium dodecyl sulfate, 0.1 g; distilled water, 100 ml), and the number of oocysts present was determined in a modified Neubauer *
Corresponding author. 3514
hemacytometer. The suspension was then diluted to the required concentration. Sewage treatment. Wastewater samples were obtained from the Santiago de Compostela municipal plant (La Corufia province). This plant receives 40,000 m of combined (domestic and industrial) wastewater per day with a biological oxygen demand of 200 to 450 mg/liter in 5 days and with about 400 mg of suspended solids per liter. The activatedsludge system employed in secondary treatment is of the plug flow type, with surface aeration by mechanical stirring (dissolved oxygen, 1.5 to 2 mg/liter). The mixed liquor (return sludge) contains 2,000 to 2,500 mg of suspended solids per liter and 70 to 80% volatile suspended substances; the sludge volume index is 50 to 125 mlIg, and the mean cell residence time is 4 to 5 days. The final effluent discharged by the plant has a biological oxygen demand of 15 to 20 mg/liter in 5 days and about 100 mg of suspended solids per liter. Samples for procedure B were of primary clarifier effluent, i.e., of wastewater which had been treated only by coarse screening, sand removal, and decantation (particles >1 mm in diameter). Samples for procedure A (control) were of final effluent, i.e., of wastewater which had received activatedsludge treatment. The activated sludge used in procedure B was obtained from the plant's mixed liquor. Laboratory treatment of effluent. For both procedures (A and B), 500-ml samples (of primary clarifier effluent or final effluent, pH 7.5) were enriched with C parvum oocysts (2.5 x 108 oocysts per liter for experiment 1 and 4.0 x 108 oocysts per liter for experiment 2, with two replications of each experiment). Procedure A samples received no further treatment prior to inoculation of mice. Procedure B samples were subjected to a laboratory simulation of the activated-sludge treatment process. Since in the treatment plant return sludge is added to incoming effluent at a ratio of between 2.8:1 and 3:1, in the laboratory simulation 1,500 ml of activated sludge was added to each 500-ml sample. The mixture was then bubbled with air for 3 h, after which it was transferred to a flask for 35 to 40 min in order to allow flocculated particles to settle. The supernatant was divided into aliquots, which were centrifuged at 1,300 x g for 5 min; the resulting pellets were mixed together again and resuspended in the supernatant to a volume of 500 ml. The mixture was then homogenized. Experimental infection. By means of a 16-mm-long, 26-
ACrIVATED-SLUDGE TREATMENT OF C PARVUM
VOL. 58, 1992
3515
TABLE 1. Effects of activated-sludge treatment on numbers of oocysts in effluent and intensity of infection in mice No. of oocysts/liter of primary clarifier effluent Expt no.
Before treatment
la lb 2a 2b
2.5 x 108 2.5 x 108 4 x 108 4 x 108
After treatment
0.43 0.40 0.80 0.80
x 108 x 108 x 108 x 108
Mean intensity of infection in mice (10 oocysts/ml of homogenized intestine)' Procedure A
B Procedure (test litters)
19.4 17.6 1,750 82.9
0.12 0.18 331 14.9
(control litters)
(100) (100) (100) (100)
(4.8) (6)
(95.6) (74)
% Reduction in infection intensity (procedure B vs
procedure A)
99 98 92 82
a Numbers in parentheses indicate percent incidence of infection in control and test litters.
gauge needle fitted with plastic tubing, litters of 9 to 15 mice aged 2 to 4 days were intragastrically inoculated with 0.1 ml of the oocyst-enriched wastewater treated as described above (procedure A or procedure B). Mice were killed by inhalation of ether 7 days postinfection. The small and large intestines were removed and placed in 5 ml of cold phosphate-buffered saline containing streptomycin (1.0 mg/ml) and penicillin (1.0 x 103 IU/ml). The total intestinal oocyst content was evaluated after homogenization of the intestines (three times, 10 s each) with an Ultra-Turrax homogenizer. Oocysts were counted in a hemacytometer as described above; if no oocysts were detected, the sample was centrifuged and the sediment was reexamined by using carbolfuchsin staining. Statistics. Mean oocyst counts were compared by using the Mann-Whitney U test (18).
RESULTS Counts of C. parvum oocysts in contaminated primary clarifier effluent (procedure B) before and after laboratory simulation of activated-sludge treatment indicated reductions in initial oocyst numbers in enriched primary clarifier effluent of 83 and 84% (experiments la and lb, respectively) and 80% (experiments 2a and 2b) (Table 1). The effect of treatment was statistically significant (P < 0.05) in all cases. In the control litters (procedure A), a 100% incidence of infection was observed in all cases. Although the oocyst enrichment of the primary effluent was increased by a factor of 1.6 from experiment 1 to experiment 2, mean reductions in the infection intensity after laboratory activated-sludge treatment (experiments la and lb versus experiments 2a and 2b) differed by only 11.5% (Table 1).
DISCUSSION Cryptosponidium oocysts have been found in natural waters in several previous studies (11, 13, 15); possible sources of contamination include urban and agricultural (especially dairy cattle) slurry, septic tank leakage, recreational bathing, agricultural runoff, and erosion of soils exposed to infected feces. About 15,000 cases of cryptosporidiosis in the United States, as well as various outbreaks of Cryptosporidiumrelated diarrheal illness in the United Kingdom, have been attributed to consumption of contaminated drinking water (10, 17). In light of these studies, C. parvum can be considered a waterborne organism with significant implications for public health. Although the activated-sludge process is not generally considered efficient in removing protozoan cysts (1), Madore et al. (11) state that about 79% of Cryptosporidium oocysts are removed from sewage treated in this way. In our study,
the rate of removal of C. parvum oocysts from sewage subjected to a laboratory simulation of activated-sludge treatment (80 to 84%) was similar to that reported by other authors. However, the fact that infection still developed in mice inoculated with sewage treated in this way (test litters) indicates that the remaining oocysts maintain their infectivity and are present in sufficient numbers for infection to occur. Indeed, the number of oocysts present in the 0.1-ml inoculation dose used exceeded the minimum infective dose established by us in previous work (14). The marked difference between the mean intensities of infection obtained in control litters might have been due to heterogeneity in the infectivity of the C parvum isolates used or in the responses of the mice to infection (2). In Spain, use of activated sludge is the standard method for wastewater treatment (7), with the treated sewage often being discharged directly to watercourses which also serve as sources of drinking water. It has been demonstrated previously that chlorination, the most commonly used water disinfection technique, is not effective in inactivating C. parvum oocysts (9). We thus conclude that additional water purification procedures are necessary if the health risk posed by this organism is to be effectively eliminated (14). ACKNOWLEDGMENTS We thank Carlos Aymerich, technical manager of the sewage treatment plant run by DEGREMONT, S. A., and German de la Iglesia, biologist with AQUAGEST, S. A., in Santiago de Compostela, La Corufna, Spain, for their skilled technical assistance and valuable contributions to this study. REFERENCES 1. Bitton, G. 1980. Introduction to environmental virology. John Wiley & Sons, Inc., New York. 2. Current, W. L., and L. S. Garcia. 1991. Cryptosporidiosis. Clin. Microbiol. Rev. 4:325-358. 3. D'Antonio, R. G., R. E. Wimn, J. P. Taylor, T. L. Gustafson, W. L. Current, M. M. Rhodes, G. W. Gary, and R. A. Zajac. 1985. A waterborne outbreak of cryptosporidiosis in normal hosts. Ann. Intern. Med. 103:886-888. 4. Gailaher, M. M., J. L. Herndon, L. J. Nims, C. R. Sterling, D. J. Grabowski, and H. F. Hull. 1989. Cryptosporidiosis and surface water. Am. J. Public Health 79:39-42. 5. Hayes, E. B., T. D. Matte, T. R. O'Brien, T. W. McKinley, G. S. Longsdon, J. B. Rose, B. L. P. Ungar, D. M. Word, P. F. Pinsky, M. L. Cummings, M. A. Wilson, E. G. Long, E. S. Hurwitz, and D. D. Juranek. 1989. Large community outbreak of cryptosporidiosis due to contamination of a filtered public water supply. N. Engl. J. Med. 320:1372-1376. 6. Heine, J. 1982. Eine einfache Nachweismethode fuir Kryptosporidien im Kot. Zentralbl. Veterinaermed. Reihe B
29:324-327.
7. Herriez, I., J. L6pez, L. Rubio, and M. E. Fernandez. 1989. Residuos urbanos y medio ambiente. Ediciones Universidad
VILLACORTA-MARTINEZ DE MATURANA ET AL.
APPL. ENVIRON. MICROBIOL.
Aut6noma, Madrid. 8. Isaac-Renton, J. L., D. Fogel, H. H. Stibbs, and J. E. Ongerth. 1987. Giardia and Cryptosporidium in drinking water. Lancet i:973-974. 9. Korich, D. G., J. R. Mead, M. S. Madore, N. A. Sinclair, and C. R. Sterling. 1990. Effects of ozone, chlorine dioxide, chlorine, and monochloramine on Cryptosporidium parvum oocyst viability. Appl. Environ. Microbiol. 56:1423-1428. 10. Longsdon, G. S., D. Juranek, L. Manson, J. E. Ongerth, J. B. Rose, C. R. Sterling, and B. L. P. Ungar. 1988. Roundtable Cryptosporidium. J. Am. Water Works Assoc. 80:14-27. 11. Madore, M. S., J. B. Rose, C. P. Gerba, M. J. Arrowood, and C. R. Sterling. 1987. Occurrence of Cryptosporidium oocysts in sewage effluents and selected surface waters. J. Parasitol. 73:702-705. 12. Musial, C. E., M. J. Arrowood, C. R. Sterling, and C. P. Gerba. 1987. Detection of Cryptosporidium in water by using polypropylene cartridge fflters. Appl. Environ. Microbiol. 53:687-692. 13. Ongerth, J. E., and H. H. Stibbs. 1987. Identification of Cryp-
tosporidium oocysts in river water. Appl. Environ. Microbiol. 53:672-676. Peeters, J. E., E. Ares Maz6s, W. J. Masschelein, I. Villacorta Martinez de Maturana, and E. Debacker. 1989. Effect of disinfection of drinking water with ozone or chlorine dioxide on survival of Cryptosporidium parvum oocysts. Appl. Environ. Microbiol. 55:1519-1522. Rose, J. B., A. Cifrino, M. S. Madore, C. P. Gerba, C. R. Sterling, and M. J. Arrowood. 1986. Detection of Cryptosporidium from wastewater and freshwater environments. Water Sci. Technol. 18:233-239. Rush, B. A., P. A. Chapman, and R. W. Ineston. 1987. Cryptosporidium and drinking water. Lancet ii:632-633. Smith, H. V., R. W. Girdwood, W. J. Patterson, R. Hardie, L. A. Green, C. Benton, W. Tulloch, J. C. Sharp, and G. I. Forbes. 1988. Waterborne outbreak of cryptosporidiosis. Lancet ii:1484. Sokal, R. R., and F. J. Rohlf. 1973. Introduction to biostatistics, p. 217-220. W. H. Freeman & Co., San Francisco.
3516
14.
15.
16. 17. 18.
Vol. 58, No. 11
Efficacy of Activated Sludge in Removing Cryptosporidium parvum Oocysts from Sewage I. VILLACORTA-MARTINEZ
DE MATURANA, M. E. ARES-MAZAS,* AND M. J. LORENZO-LORENZO
D. DURAN-OREIRO,
Laboratory of Parasitology, Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Santiago de Compostela, 15706 Santiago de Compostela, La Corufia, Spain Received 25 February 1992/Accepted 1 September 1992
Primary clarifier effluent (procedure B) and final effluent (procedure A) from a wastewater treatment plant enriched with Cryptosporidium parvum oocysts obtained from the feces of naturally infected calves. Procedure B samples alone were subjected to a laboratory simulation of activated-sludge treatment. Coccidium-free neonatal CD-1 mice were then inoculated intragastrically with procedure A or procedure B samples. Seven days after inoculation, the intensity of oocyst infection in procedure B mice was 91% less than in procedure A mice (controls).
were
Cryptosporidium parvum (Apicomplexa, Cryptosporidiidae) is a monoxenous coccidian protozoan parasite causing diarrheal disease in a wide range of vertebrates including humans, with serious consequences in neonates and immunodeficient individuals. Recent reports of cryptosporidiosis outbreaks suggest that contamination of water supplies plays an important role in transmitting the disease (3-5, 8, 16), a hypothesis supported by the detection of oocysts of both human and animal origin in surface waters and sewage (11-13, 15). Furthermore, it has been shown that disinfectants for drinking water purification, with the possible exception of ozone (9, 14), are not very effective against C. parvum. In this study, we evaluated the efficacy of a sewage treatment system (activated sludge and flocculation) in removing C. parvum oocysts from contaminated sewage by using a laboratory simulation of the treatment process. MATERIALS AND METHODS Animals and husbandry. The 12 litters of coccidium-free neonatal CD-1 mice used were kept separately with their dams at 20°C in Panlab plastic cages with wire mesh tops and wood shavings for bedding and received a commercial pelleted feed (Letica, Barcelona, Spain) and water ad libitum.
Oocysts. One- to two-week-old Friesian calves were individually screened for excretion of C parvum oocysts by carbolfuchsin staining (6). Fresh fecal material was collected by rectal sampling, immediately suspended 1:1 in cold 5% (wt/vol) aqueous potassium dichromate solution, and stored at 4°C until use. After several washes with demineralized water to eliminate potassium dichromate, the feces were washed through a 45-,um-pore-size mesh and centrifuged several times in demineralized water-diethyl ether at 1,000 x g for 5 min; the resulting concentrated fluid was washed again to eliminate the diethyl ether. For counting, 0.2 ml of the suspension was mixed with 0.8 ml of a malachite green preparation (malachite green, 0.16 g; sodium dodecyl sulfate, 0.1 g; distilled water, 100 ml), and the number of oocysts present was determined in a modified Neubauer *
Corresponding author. 3514
hemacytometer. The suspension was then diluted to the required concentration. Sewage treatment. Wastewater samples were obtained from the Santiago de Compostela municipal plant (La Corufia province). This plant receives 40,000 m of combined (domestic and industrial) wastewater per day with a biological oxygen demand of 200 to 450 mg/liter in 5 days and with about 400 mg of suspended solids per liter. The activatedsludge system employed in secondary treatment is of the plug flow type, with surface aeration by mechanical stirring (dissolved oxygen, 1.5 to 2 mg/liter). The mixed liquor (return sludge) contains 2,000 to 2,500 mg of suspended solids per liter and 70 to 80% volatile suspended substances; the sludge volume index is 50 to 125 mlIg, and the mean cell residence time is 4 to 5 days. The final effluent discharged by the plant has a biological oxygen demand of 15 to 20 mg/liter in 5 days and about 100 mg of suspended solids per liter. Samples for procedure B were of primary clarifier effluent, i.e., of wastewater which had been treated only by coarse screening, sand removal, and decantation (particles >1 mm in diameter). Samples for procedure A (control) were of final effluent, i.e., of wastewater which had received activatedsludge treatment. The activated sludge used in procedure B was obtained from the plant's mixed liquor. Laboratory treatment of effluent. For both procedures (A and B), 500-ml samples (of primary clarifier effluent or final effluent, pH 7.5) were enriched with C parvum oocysts (2.5 x 108 oocysts per liter for experiment 1 and 4.0 x 108 oocysts per liter for experiment 2, with two replications of each experiment). Procedure A samples received no further treatment prior to inoculation of mice. Procedure B samples were subjected to a laboratory simulation of the activated-sludge treatment process. Since in the treatment plant return sludge is added to incoming effluent at a ratio of between 2.8:1 and 3:1, in the laboratory simulation 1,500 ml of activated sludge was added to each 500-ml sample. The mixture was then bubbled with air for 3 h, after which it was transferred to a flask for 35 to 40 min in order to allow flocculated particles to settle. The supernatant was divided into aliquots, which were centrifuged at 1,300 x g for 5 min; the resulting pellets were mixed together again and resuspended in the supernatant to a volume of 500 ml. The mixture was then homogenized. Experimental infection. By means of a 16-mm-long, 26-
ACrIVATED-SLUDGE TREATMENT OF C PARVUM
VOL. 58, 1992
3515
TABLE 1. Effects of activated-sludge treatment on numbers of oocysts in effluent and intensity of infection in mice No. of oocysts/liter of primary clarifier effluent Expt no.
Before treatment
la lb 2a 2b
2.5 x 108 2.5 x 108 4 x 108 4 x 108
After treatment
0.43 0.40 0.80 0.80
x 108 x 108 x 108 x 108
Mean intensity of infection in mice (10 oocysts/ml of homogenized intestine)' Procedure A
B Procedure (test litters)
19.4 17.6 1,750 82.9
0.12 0.18 331 14.9
(control litters)
(100) (100) (100) (100)
(4.8) (6)
(95.6) (74)
% Reduction in infection intensity (procedure B vs
procedure A)
99 98 92 82
a Numbers in parentheses indicate percent incidence of infection in control and test litters.
gauge needle fitted with plastic tubing, litters of 9 to 15 mice aged 2 to 4 days were intragastrically inoculated with 0.1 ml of the oocyst-enriched wastewater treated as described above (procedure A or procedure B). Mice were killed by inhalation of ether 7 days postinfection. The small and large intestines were removed and placed in 5 ml of cold phosphate-buffered saline containing streptomycin (1.0 mg/ml) and penicillin (1.0 x 103 IU/ml). The total intestinal oocyst content was evaluated after homogenization of the intestines (three times, 10 s each) with an Ultra-Turrax homogenizer. Oocysts were counted in a hemacytometer as described above; if no oocysts were detected, the sample was centrifuged and the sediment was reexamined by using carbolfuchsin staining. Statistics. Mean oocyst counts were compared by using the Mann-Whitney U test (18).
RESULTS Counts of C. parvum oocysts in contaminated primary clarifier effluent (procedure B) before and after laboratory simulation of activated-sludge treatment indicated reductions in initial oocyst numbers in enriched primary clarifier effluent of 83 and 84% (experiments la and lb, respectively) and 80% (experiments 2a and 2b) (Table 1). The effect of treatment was statistically significant (P < 0.05) in all cases. In the control litters (procedure A), a 100% incidence of infection was observed in all cases. Although the oocyst enrichment of the primary effluent was increased by a factor of 1.6 from experiment 1 to experiment 2, mean reductions in the infection intensity after laboratory activated-sludge treatment (experiments la and lb versus experiments 2a and 2b) differed by only 11.5% (Table 1).
DISCUSSION Cryptosponidium oocysts have been found in natural waters in several previous studies (11, 13, 15); possible sources of contamination include urban and agricultural (especially dairy cattle) slurry, septic tank leakage, recreational bathing, agricultural runoff, and erosion of soils exposed to infected feces. About 15,000 cases of cryptosporidiosis in the United States, as well as various outbreaks of Cryptosporidiumrelated diarrheal illness in the United Kingdom, have been attributed to consumption of contaminated drinking water (10, 17). In light of these studies, C. parvum can be considered a waterborne organism with significant implications for public health. Although the activated-sludge process is not generally considered efficient in removing protozoan cysts (1), Madore et al. (11) state that about 79% of Cryptosporidium oocysts are removed from sewage treated in this way. In our study,
the rate of removal of C. parvum oocysts from sewage subjected to a laboratory simulation of activated-sludge treatment (80 to 84%) was similar to that reported by other authors. However, the fact that infection still developed in mice inoculated with sewage treated in this way (test litters) indicates that the remaining oocysts maintain their infectivity and are present in sufficient numbers for infection to occur. Indeed, the number of oocysts present in the 0.1-ml inoculation dose used exceeded the minimum infective dose established by us in previous work (14). The marked difference between the mean intensities of infection obtained in control litters might have been due to heterogeneity in the infectivity of the C parvum isolates used or in the responses of the mice to infection (2). In Spain, use of activated sludge is the standard method for wastewater treatment (7), with the treated sewage often being discharged directly to watercourses which also serve as sources of drinking water. It has been demonstrated previously that chlorination, the most commonly used water disinfection technique, is not effective in inactivating C. parvum oocysts (9). We thus conclude that additional water purification procedures are necessary if the health risk posed by this organism is to be effectively eliminated (14). ACKNOWLEDGMENTS We thank Carlos Aymerich, technical manager of the sewage treatment plant run by DEGREMONT, S. A., and German de la Iglesia, biologist with AQUAGEST, S. A., in Santiago de Compostela, La Corufna, Spain, for their skilled technical assistance and valuable contributions to this study. REFERENCES 1. Bitton, G. 1980. Introduction to environmental virology. John Wiley & Sons, Inc., New York. 2. Current, W. L., and L. S. Garcia. 1991. Cryptosporidiosis. Clin. Microbiol. Rev. 4:325-358. 3. D'Antonio, R. G., R. E. Wimn, J. P. Taylor, T. L. Gustafson, W. L. Current, M. M. Rhodes, G. W. Gary, and R. A. Zajac. 1985. A waterborne outbreak of cryptosporidiosis in normal hosts. Ann. Intern. Med. 103:886-888. 4. Gailaher, M. M., J. L. Herndon, L. J. Nims, C. R. Sterling, D. J. Grabowski, and H. F. Hull. 1989. Cryptosporidiosis and surface water. Am. J. Public Health 79:39-42. 5. Hayes, E. B., T. D. Matte, T. R. O'Brien, T. W. McKinley, G. S. Longsdon, J. B. Rose, B. L. P. Ungar, D. M. Word, P. F. Pinsky, M. L. Cummings, M. A. Wilson, E. G. Long, E. S. Hurwitz, and D. D. Juranek. 1989. Large community outbreak of cryptosporidiosis due to contamination of a filtered public water supply. N. Engl. J. Med. 320:1372-1376. 6. Heine, J. 1982. Eine einfache Nachweismethode fuir Kryptosporidien im Kot. Zentralbl. Veterinaermed. Reihe B
29:324-327.
7. Herriez, I., J. L6pez, L. Rubio, and M. E. Fernandez. 1989. Residuos urbanos y medio ambiente. Ediciones Universidad
VILLACORTA-MARTINEZ DE MATURANA ET AL.
APPL. ENVIRON. MICROBIOL.
Aut6noma, Madrid. 8. Isaac-Renton, J. L., D. Fogel, H. H. Stibbs, and J. E. Ongerth. 1987. Giardia and Cryptosporidium in drinking water. Lancet i:973-974. 9. Korich, D. G., J. R. Mead, M. S. Madore, N. A. Sinclair, and C. R. Sterling. 1990. Effects of ozone, chlorine dioxide, chlorine, and monochloramine on Cryptosporidium parvum oocyst viability. Appl. Environ. Microbiol. 56:1423-1428. 10. Longsdon, G. S., D. Juranek, L. Manson, J. E. Ongerth, J. B. Rose, C. R. Sterling, and B. L. P. Ungar. 1988. Roundtable Cryptosporidium. J. Am. Water Works Assoc. 80:14-27. 11. Madore, M. S., J. B. Rose, C. P. Gerba, M. J. Arrowood, and C. R. Sterling. 1987. Occurrence of Cryptosporidium oocysts in sewage effluents and selected surface waters. J. Parasitol. 73:702-705. 12. Musial, C. E., M. J. Arrowood, C. R. Sterling, and C. P. Gerba. 1987. Detection of Cryptosporidium in water by using polypropylene cartridge fflters. Appl. Environ. Microbiol. 53:687-692. 13. Ongerth, J. E., and H. H. Stibbs. 1987. Identification of Cryp-
tosporidium oocysts in river water. Appl. Environ. Microbiol. 53:672-676. Peeters, J. E., E. Ares Maz6s, W. J. Masschelein, I. Villacorta Martinez de Maturana, and E. Debacker. 1989. Effect of disinfection of drinking water with ozone or chlorine dioxide on survival of Cryptosporidium parvum oocysts. Appl. Environ. Microbiol. 55:1519-1522. Rose, J. B., A. Cifrino, M. S. Madore, C. P. Gerba, C. R. Sterling, and M. J. Arrowood. 1986. Detection of Cryptosporidium from wastewater and freshwater environments. Water Sci. Technol. 18:233-239. Rush, B. A., P. A. Chapman, and R. W. Ineston. 1987. Cryptosporidium and drinking water. Lancet ii:632-633. Smith, H. V., R. W. Girdwood, W. J. Patterson, R. Hardie, L. A. Green, C. Benton, W. Tulloch, J. C. Sharp, and G. I. Forbes. 1988. Waterborne outbreak of cryptosporidiosis. Lancet ii:1484. Sokal, R. R., and F. J. Rohlf. 1973. Introduction to biostatistics, p. 217-220. W. H. Freeman & Co., San Francisco.
3516
14.
15.
16. 17. 18.
