C. Maya*, N. Beltrán*, B. Jiménez* and P. Bonilla** * Instituto de Ingeniería, UNAM. Apartado Postal 70-472. Coyoacán, 04510. México D. F. México (E-mail:
[email protected]) ** Facultad de Estudios Superiores Iztacala. División de Investigación y Posgrado. Av. de los Barrios s/n Los Reyes Iztacala, Tlalnepantla, C.P. 54090, Estado de México, México Abstract Every year around 3.4 million people die from water-related diseases, mainly amoebiasis and diarrhoea caused by bacteria. The assessment of the efficiency of a UV light disinfection process in the inactivation of indicators, pathogen bacteria and amphizoic amoebae in a secondary treated effluent was carried out. Wastewater was irradiated with different doses of UV light using a collimated-beam reactor. Dose-response results showed that a UV dose of 15 mW·s/cm2 was enough to inactivate FC to the limit established in Mexican legislation (4 2–3 2–3
Linden et al. (2002) Craik et al. (2000) Craik et al. (2001)
Jiménez and Beltrán (2002) Sommer et al. (1998)
Quantification of bacteria
The quantification of FC, faecal streptococci and Salmonella typhi, before and after each radiation, was carried out using the membrane filtration technique in accordance with the Standard Methods (APHA, AWWA, WPCF, 1995). Salmonella typhi was quantified in pre-enrichment and enrichment broths, with confirmation of presumed positive colonies in selective agar and then in API-20E (BioMerieux).
The isolation, identification, viability and pathogenicity of amphizoic amoebae were carried out in accordance with de Jonckheere (1977). For the qualitative studies the monoxenic medium was used (NNE) consisting of non-nutrient agar broth with the addition of inactivated Enterobacter aerogenes (which provides the salts and the source of carbon, in the form of bacteria, necessary for its support) and the axenic liquid medium (PBSGM, Chang, modified) composed of phosphate, biotriptase, fetal calf serum, glucose and, penicillin and kanamicine added at a concentration of 200 µg/ml of each, which is used to isolate and maintain the strains that have grown in the monoxenic medium. The identification of the trophic and cystic forms was carried out using a phase contrast microscope with 20 × and 40 × objectives and in accordance with Page (1988). The general conditions of quantification, isolation, identification and culture used for each microorganism are summarized in Table 2.
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Isolation and identification of amoebae
Preparation of the amoebae suspension
The amoebae in the trophozoite form were inoculated into the axenic medium and incubated at 30°C. for later concentration. The incubated medium was centrifuged at 2,500 rpm/10 min and the quantification was carried out using a Neubauer chamber for hematocits with a depth of 0.1 mm. This procedure was repeated until it was reached a trophozoites concentration of 105 cells/ml of medium, as long as the medium edge was not more than 7 days. Pathogenicity test
Two groups of five white mice, males of the Mus musculus species strain CD-1, 3 weeks old, were inoculated with 0.02 ml of the amoebae suspension. One of the groups was inoculated intracerebrally and the other one intranasally. A parallel group was inoculated with an amoeba-free culture medium as a control group. The mice were checked daily to observe changes in behavior, vital signs and the periodicity and number of deaths. Once the mice died or completed the trial period (21 days) a necropsy was performed to examine the damage caused by the amoebae.
Table 2 General conditions for isolation, identification and microbiological culture Microorganisms
Bacteria FC Faecal streptococci Salmonella typhi Amoebae Acanthamoeba spp.
N.A. Not applicable M.F. Membrane filtration
Reference strain
Growth medium
Incubation conditions
Technique
N.A. N.A. Salmonella typhi ATCC 6539
MFC agar (Difco) KF agar (BBL) Sulfite and Bismute agar (Bioxon)
44.5°C, 18–24 h 35°C, 48 h 35°C, 18–24 h
M.F. M.F. M.F.
Acanthamoeba culbertsoni ATCC 30171
Monoxenic, NNE Axenic, PBSGM
30–35°C, 3–4 weeks 35°C, 1–2 weeks
Microscopy
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UV lamp
Collimating tube
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Shutter Radiometer Sample in Petri dish
Magnetic stirrer
Figure 1 Typical diagram for a collimated beam equipment Radiation tests
The disinfection tests were carried out using a collimated-beam reactor (reactor used to project light rays perpendicularly onto the surface of the sample) donated by Trojan Technologies Inc., which consists of a low pressure mercury lamp suspended horizontally (Figure 1). The samples of wastewater in aliquots of 50 ml were irradiated in 55 mm diameter sterile containers and stirred continuously with 25 mm magnetic bars. In the specific case of the amoebae, the irradiated sample was prepared from the suspension and previously filtered and sterilized water from the secondary effluent. The concentration of the reference strain of amoeba Acanthamoeba culbertsoni ATTC 30171 and of the isolated strain Acanthamoeba spp. was approximately 1.6 × 104 trophozoites. Almost immediately, the samples were retrieved by centrifugation (at 2,500 rpm/15 min), quantified and inoculated onto NNE medium to be incubated at 30°C. They were observed daily by microscope to determine the inactivation efficiency (viability) and increase or decrease the contact time as necessary. Dose determination
The intensity of the UV light was measured with an International Light Inc. calibrated radiometer, IL 1700 with a SED240 #4817 detector, which measures a band width of between 200 and 320 nm. The detector is placed at the same elevation as the surface of the sample to be irradiated and the measured intensity is corrected with the absorption of liquid in accordance with Beer’s law (Morowitz, 1950). The dose (mW·s/cm2) is the result of the corrected intensity (mW/cm2) and the exposure (s) time. Results and discussion Bacteria inactivation
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The inactivation of FC with a maximum applied dose of 60 mW·s/cm2 was 5 log units (Figure 2). However, a dose of 15 mW·s/cm? is sufficient to comply with the Official Mexican Standard (NOM-001-ECOL, 1996), which establishes a maximum permissible limit of 1,000 MPN/100 ml for reuse for agricultural irrigation. Although faecal streptococci are not considered to be indicators of pollution in Mexican legislation, in this study its degree of resistance was evaluated in comparison with FC. The inactivation kinetics of
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these shows a lag phase that is observed when applying doses lower than 15 mW·s/cm2. This behavior is attributed to the size of these microorganisms (1 µm), they are smaller than other bacteria (FC and Salmonella typhi 0.5 × 2–5 µm), which is why they are not considered an easy target to attack with UV light. It is also attributed to the clusters formed by these bacteria (in chains), which prevent rapid inactivation. However, even though this lag phase occurs in low doses, it is possible to inactivate up to 6 log applying a 60 mW·s/cm2 dose. The Salmonella typhi inactivation results (Figure 2) show that when a dose of approximately 30 mW·s/cm2 is applied, a reduction of up to 3 log units can be achieved with initial and final concentrations of 4 × 104 MPN/100 ml and 43 MPN/100 ml respectively. With this dose a minimum risk of infection by this pathogen is assured (concentrations 20 min) and doses higher than 173 mW·s/cm2 (Figure 3) for their total inactivation. This can be explained by the presence of walls on the cysts, secreted as a kind of “extracellular armour” whose phosphoprotein (external) and cellulose (internal) consistency gives them 6
Inactivation (log)
5
4
3
2 Fa e ca l c olifo rms S a lmo n el la t yp h i
1
Fa e ca l e s tre pt o co cc i 0 0
10
20
30
40
50
60
70
Dose (mW·s/cm2)
Figure 2 Bacteria inactivation Table 3 Percentage of Acanthamoeba spp. pathogenic in mice Strain
Acanthamoebaspp.
Pathogenicity (%) Death time (d)
100 3–5
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6 Acanthamoeba culbersoni
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Trophozoites inactivation (log)
5
Acanthamoeba spp.
4 3 2 1 0 0
20
40
60
80
100
120
140
160
180
200
Dose (mW·s/cm2)
Figure 3 Amphizoic amoebae inactivation
a
b
Figure 4 Cysts (a) and trophozoites (b) of Acanthamoeba spp. (Nomarski technique) 40 × 1 (photographs taken in colaboration with Dr. Miroslav Macek, FES-Iztacala, UNAM)
great resistance. Nevertheless, the dose required to achieve a 2 log inactivation (60 mW·s/cm2) is comparable to those reported necessary to achieve the same degree of inactivation of other protozoa. Cysts and trophozoites of the isolated strain of Acanthamoeba spp. are shown in Figure 4. Conclusions
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The results show that a dose of 15 mW·s/cm2 is sufficient to reduce the concentration of FC to concentrations lower than those established by Mexican legislation for reuse of treated wastewater for agricultural irrigation. In addition, with that dose it is possible to inactivate Salmonella typhi to concentrations that are not sufficient to cause infection (less than 103 MPN) and reduce faecal streptococci to 5 log, which represents a final concentration of 100 MPN/100 ml. In the case of the free-living amoebae, it is demonstrated that the doses needed to inactivate them are comparable to those reported for other kinds of microorganisms such as Giardia muris and Cryptosporidium parvum, approximately 60 mW·s/cm2 to achieve inactivation between 2 and 3 log. However, with doses of 173 mW·s/cm2 it was possible to inactivate up to 5 log. These results show the possibility of using UV light as an alternative to disinfection with chlorine as well as considering alternative groups of microbiological pollution indicators within the present normativity for the reuse of treated wastewater, principally in the
countries where the presence of pathogen microorganisms represents a serious public health problem. Besides, additional testing would be convenient to establish necessary doses for the inactivation of cysts and trophozoites of different species of amoebae. Acknowledgements
References APHA, AWWA, WPCF (1995). Standard Methods for the Examination of Water and Wastewater. American Public Health Association, Washington DC., USA. Cairns, W.L., Sakamoto, G., Comair, C.B. and Gehr, R. (1993). Assessing UV Disinfection of a PhysicoChemical Effluent by Medium Pressure Lamps Using a Collimated Beam and Pilot Plant. Trojan Technologies Inc. 3020 Gore Road, London, Ontario Canada N5V 4T7. Conde-Bonfil, M.C. and De la Mora-Zerpa, C. (1992). Entamoeba histolytica: A standing threat. Mexico Public Health Journal, 34(3), 335–341 (in Spanish). Craik, S., Finch, G., Bolton, J. and Belosevic, M. (2000). Inactivation of Giardia muris cysts using mediumpressure ultraviolet radiation in filtered drinking water. Wat. Res., 34(18), 4325–4332. Craik, S., Weldon, D., Finch, G., Bolton, J. and Belosevic, M. (2001). Inactivation of Cryptosporidium parvum oocysts using medium and low-pressure ultraviolet radiation. Wat. Res., 35(6), 1387–1398. Craun, G.F. (1988). Surface water supplies and health. J. Am. Wat. Waste Assoc., 80, 40–52. De Jonckheere, J.F. (1977). Use of an axenic medium for differentiation between pathogenic and non pathogenic Naegleria fowleri isolates. Appl. Env. Microb., 33, 751–757. Jiménez, B.E., Maya, C. and Salgado, G. (2001). The elimination of helminth ova, faecal coliforms, Salmonella and protozoan cysts by various physicochemical processes in wastewater and sludge. Wat. Sci. Tech., 43(12), 179–182. Jiménez, B. and Beltrán, N. (2002). Efficiency of UV light disinfection in wastewater with high content of pathogens. Health-Related Water Microbiology Symposium. IWA 3rd World Water Congress. Melbourne, Australia, 7–12 April. Kool, H.J., Kreijl, C.F. and Hrubec, J. (1985). Mutagenic and carcinogenic properties of drinking water in water chlorination. R.L. Jolley, R.J. Bull, W.P. Davis, S. Katz, M.H., Roberts, Jr., and V.A. Jacobs (Eds.). Chemistry, Environmental Impact and Health Effects, Lewis Publishers, Chelsea, Michigan, USA, 5,187–205. Lazarova, V., Janex, M., Fiksdal, L., Oberg, C., Barcina, I. and Pommepuy, M. (1998). Advanced wastewater disinfection technologies: short and long term efficiency. Wat. Sci. Tech., 38(12), 109–117. Lazarova, V., Savoye, P., Janex, M., Blatchley, E. and Pommepuy, M. (1999). Advanced wastewater disinfection technologies: state of the art and perspectives. Wat. Sci. Tech., 40(4–5), 203–213. Linden, K., Shin, G., Faubert, G., Cairns, W. and Sobsey, M. (2002). UV disinfection of Giardia lamblia cysts in water. Env. Sci. Tech., 36, 2519–2522. Martínez, A.J. and Visvesvara, G.S. (1997). Free-living, amphizoic and opportunistic amebas. Brain Pathol., 7, 583–589. Morowitz, H. (1950). Absorption effects in volume irradiation of microorganisms. Sci., 111, 229–230. NOM-001-ECOL (1996). Official Mexican Legislative. Norms which Establish the Maximum Levels of Contaminants in Wastewater Discharges. Diario Oficial de la Federación, City of Mexico, Mexico 6 January, 67–81. Page, F.C. (1988). A New Key to Freshwater and Soil Gymnamoebae. Freshwater Biological Association Scientific Publication England, UK, p. 122. Shin, G., Linden, K. and Sobsey, M. (2000). Comparative inactivation of Cryptosporidium parvum oocysts and coliphage MS2 by monochromatic UV irradiation. Disinfection 2000: Disinfection of Wastes in the New Millenium. New Orleans, LA., USA. March 15–18. Sommer, R., Haider, T., Cabaj, A., Pribil, W. and Lhotsky, M. (1998). Time dose reciprocity in UV disinfection of water. Wat. Sci. Tech., 38(12), 145–150. US.EPA (1999). Alternative disinfectants and oxidants. US.EPA Guidance Manual, Cincinnati, Ohio, USA. WHO (2002). World Health Report 2002. Reducing risks, promoting healthy life.
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The authors thank Trojan Technologies INC for its technical and financial support for this project.
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